--- OA_place: publisher OA_type: hybrid _id: '22148' abstract: - lang: eng text: 'How the twin-arginine translocase (Tat) system transports fully folded substrate proteins across cellular membranes without disrupting membrane integrity has been a fundamental question in cell biology for decades. The Tat system, found in prokaryotes and plant organelles, recognizes a cargo signal peptide via a conserved twin-arginine motif. The multi-subunit Tat complex facilitates the proton-motive-force-dependent translocation process, yet its overall architecture has remained unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure of the Escherichia coli (E. coli) trimeric TatB₃C₃ complex with bound substrate SufI, assembled in vivo. The complex adopts an unusual, wide-open, bowl-shaped architecture with a polar inner cavity. Unexpectedly, the cargo is engaged in a dual-contact mode: while the signal peptide binds inside one TatBC unit, the folded domain docks tightly onto an adjacent unit, possibly performing a proofreading function. This structure provides a mechanistic framework for substrate engagement and suggests the direct involvement of the entire Tat complex in substrate translocation.' acknowledged_ssus: - _id: EM-Fac - _id: ScienComp acknowledgement: We thank IST Austria for providing the funding. We thank IST Austria EM facility for the use of Titan Krios TEM. Data processing was performed using IST high-performance computer cluster. We thank Dr. R. Roemhild and Professor C. Guet (ISTA) for help in constructing Tat deletion strains and Dr. A. Charnagalov (ISTA) for technical help. article_processing_charge: Yes (via OA deal) article_type: original author: - first_name: Ziyu full_name: Zhao, Ziyu id: a63fe682-9f3a-11ee-bf8c-cfdf919b9850 last_name: Zhao - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 biorxivid: 1 citation: ama: Zhao Z, Sazanov LA. Structure of E. Coli twin-arginine translocase (Tat) complex with bound cargo. Molecular Cell. doi:10.1016/j.molcel.2026.05.026 apa: Zhao, Z., & Sazanov, L. A. (n.d.). Structure of E. Coli twin-arginine translocase (Tat) complex with bound cargo. Molecular Cell. Elsevier. https://doi.org/10.1016/j.molcel.2026.05.026 chicago: Zhao, Ziyu, and Leonid A Sazanov. “Structure of E. Coli Twin-Arginine Translocase (Tat) Complex with Bound Cargo.” Molecular Cell. Elsevier, n.d. https://doi.org/10.1016/j.molcel.2026.05.026. ieee: Z. Zhao and L. A. Sazanov, “Structure of E. Coli twin-arginine translocase (Tat) complex with bound cargo,” Molecular Cell. Elsevier. ista: Zhao Z, Sazanov LA. Structure of E. Coli twin-arginine translocase (Tat) complex with bound cargo. Molecular Cell. mla: Zhao, Ziyu, and Leonid A. Sazanov. “Structure of E. Coli Twin-Arginine Translocase (Tat) Complex with Bound Cargo.” Molecular Cell, Elsevier, doi:10.1016/j.molcel.2026.05.026. short: Z. Zhao, L.A. Sazanov, Molecular Cell (n.d.). corr_author: '1' das_tickbox: '1' dataavailabilitystatement: "This study did not generate new unique reagents. Strains and plasmids generated in this study are available from the lead contact without restrictions.\r\n• Source data are provided within this paper. The cryo-EM map is deposited in the Electron Microscopy Data Bank under accession number EMD-53848. The model is deposited in the Protein Data Bank under accession number 9R91. The structural data are publicly available as of the date of publication. Raw images of spot assays, SDS-PAGE and BN-PAGE gels with Coomassie staining and immunoblot images are available at Mendeley Data (https://doi.org/10.17632/v2g3p9n985.1).\r\n• This paper does not report original code.\r\n• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request." date_created: 2026-06-28T22:01:35Z date_published: 2026-06-22T00:00:00Z date_updated: 2026-08-04T09:27:06Z day: '22' ddc: - '570' department: - _id: LeSa doi: 10.1016/j.molcel.2026.05.026 external_id: biorxivid: - 10.1101/2025.09.16.676506 has_accepted_license: '1' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1016/j.molcel.2026.05.026 month: '06' oa: 1 oa_version: Published Version publication: Molecular Cell publication_identifier: eissn: - 1097-4164 issn: - 1097-2765 publication_status: inpress publisher: Elsevier quality_controlled: '1' related_material: link: - description: News on ISTA website relation: press_release url: https://ista.ac.at/en/news/the-gate-for-bulky-cargo/ record: - id: '22189' relation: research_data status: for_moderation researchdata_availability: yes scopus_import: '1' status: public supplementarymaterial: yes title: Structure of E. Coli twin-arginine translocase (Tat) complex with bound cargo tmp: image: /images/cc_by_nc.png legal_code_url: https://creativecommons.org/licenses/by-nc/4.0/legalcode name: Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) short: CC BY-NC (4.0) type: journal_article user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2026' ... --- DOAJ_listed: '1' OA_place: publisher OA_type: gold PlanS_conform: '1' _id: '18987' abstract: - lang: eng text: Biallelic variants in NADH (nicotinamide adenine dinucleotide (NAD) + hydrogen (H))-ubiquinone oxidoreductase 1 alpha subcomplex 13 have been linked to mitochondrial complex I deficiency, nuclear type 28, based on three affected individuals from two families. With only two families reported, the clinical and molecular spectrum of NADH-ubiquinone oxidoreductase 1 alpha subcomplex 13–related diseases remains unclear. We report 10 additional affected individuals from nine independent families, identifying four missense variants (including recurrent c.170G > A) and three ultra-rare or novel predicted loss-of-function biallelic variants. Updated clinical–radiological data from previously reported families and a literature review compiling clinical features of all reported patients with isolated complex I deficiency caused by 43 genes encoding complex I subunits and assembly factors are also provided. Our cohort (mean age 7.8 ± 5.4 years; range 2.5–18) predominantly presented a moderate-to-severe neurodevelopmental syndrome with oculomotor abnormalities (84%), spasticity/hypertonia (83%), hypotonia (69%), cerebellar ataxia (66%), movement disorders (58%) and epilepsy (46%). Neuroimaging revealed bilateral symmetric T2 hyperintense substantia nigra lesions (91.6%) and optic nerve atrophy (66.6%). Protein modeling suggests missense variants destabilize a critical junction between the hydrophilic and membrane arms of complex I. Fibroblasts from two patients showed reduced complex I activity and compensatory complex IV activity increase. This study characterizes NADH-ubiquinone oxidoreductase 1 alpha subcomplex 13–related disease in 13 individuals, highlighting genotype–phenotype correlations. acknowledgement: "We thank all individuals and relatives for consent to be part of the study. Families 1–4, 7, were collected as part of the SYNaPS Study Group collaboration funded by The Wellcome Trust and strategic award (Synaptopathies) funding (WT093205 MA and WT104033AIA), and research was conducted as part of the Queen Square Genomics group at the University College London, supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. We are also grateful to Queen Square Genomics at the Institute of Neurology University College London, supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre, for the bioinformatics support. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.\r\nThis study was funded by the Medical Research Council (MR/S01165X/1, MR/S005021/1, G0601943). The Medical Research Council (MR/S01165X/1, MR/S005021/1, MRC ICGNMD), Wellcome Trust 221951/Z/20/Z, Global Parkinson’s Genetics Program, Aligning Science Across Parkinson’s, The Michael J. Fox Foundation, The National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetree Trust, Multiple System Atrophy Trust, Brain Research UK, Sparks Great Ormond Street Hospital Charity, Muscular Dystrophy, Muscular Dystrophy Association United States of America, and King Baudouin Foundation. H.T. was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 608473. M.S.A.-H. is funded by the Science and Technology Development Fund Academy of Science Research and Technology Egypt (Grant number: 33492, Ethical approval number: 20066). R.W.T. is funded by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), the Mitochondrial Disease Patient Cohort (UK) (G0800674), the Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), the Medical Research Council (MR/W019027/1), the Lily Foundation, Mito Foundation, the Pathological Society, LifeArc, the UK National Institute for Health Research Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children. H.H. and R.K. are supported by Global Parkinson’s Genetic Program and The Michael J. Fox Foundation Grant ID: MJFF-022153." article_number: fcae453 article_processing_charge: Yes article_type: original author: - first_name: Rauan full_name: Kaiyrzhanov, Rauan last_name: Kaiyrzhanov - first_name: Kyle full_name: Thompson, Kyle last_name: Thompson - first_name: Stephanie full_name: Efthymiou, Stephanie last_name: Efthymiou - first_name: Askhat full_name: Mukushev, Askhat last_name: Mukushev - first_name: Akbota full_name: Zharylkassyn, Akbota last_name: Zharylkassyn - first_name: Chitra full_name: Prasad, Chitra last_name: Prasad - first_name: Ehsan Ghayoor full_name: Karimiani, Ehsan Ghayoor last_name: Karimiani - first_name: Javeria Raza full_name: Alvi, Javeria Raza last_name: Alvi - first_name: Dmitriy full_name: Niyazov, Dmitriy last_name: Niyazov - first_name: Ahmad full_name: Alahmad, Ahmad last_name: Alahmad - first_name: Meisam full_name: Babaei, Meisam last_name: Babaei - first_name: Homa full_name: Tajsharghi, Homa last_name: Tajsharghi - first_name: Buthaina full_name: Albash, Buthaina last_name: Albash - first_name: Ahmad full_name: Alaqeel, Ahmad last_name: Alaqeel - first_name: Majida full_name: Charif, Majida last_name: Charif - first_name: Narges full_name: Hashemi, Narges last_name: Hashemi - first_name: Morteza full_name: Heidari, Morteza last_name: Heidari - first_name: Seyed Mehdi full_name: Kalantar, Seyed Mehdi last_name: Kalantar - first_name: Guy full_name: Lenaers, Guy last_name: Lenaers - first_name: Mohammad Yahya Vahidi full_name: Mehrjardi, Mohammad Yahya Vahidi last_name: Mehrjardi - first_name: Varunvenkat M. full_name: Srinivasan, Varunvenkat M. last_name: Srinivasan - first_name: Vykuntaraju K. full_name: Gowda, Vykuntaraju K. last_name: Gowda - first_name: Seyed Hamidreza full_name: Mirabutalebi, Seyed Hamidreza last_name: Mirabutalebi - first_name: Deanna Alexis full_name: Carere, Deanna Alexis last_name: Carere - first_name: Mojtaba full_name: Movahedinia, Mojtaba last_name: Movahedinia - first_name: David full_name: Murphy, David last_name: Murphy - first_name: Robert full_name: Mcfarland, Robert last_name: Mcfarland - first_name: Mohamed S. full_name: Abdel-Hamid, Mohamed S. last_name: Abdel-Hamid - first_name: Rasha M. full_name: Elhossini, Rasha M. last_name: Elhossini - first_name: Shahryar full_name: Alavi, Shahryar last_name: Alavi - first_name: Melanie full_name: Napier, Melanie last_name: Napier - first_name: Amaya full_name: Belanger-Quintana, Amaya last_name: Belanger-Quintana - first_name: Asuri N. full_name: Prasad, Asuri N. last_name: Prasad - first_name: Jessica full_name: Jakobczyk, Jessica last_name: Jakobczyk - first_name: Agathe full_name: Roubertie, Agathe last_name: Roubertie - first_name: Tony full_name: Rupar, Tony last_name: Rupar - first_name: Tipu full_name: Sultan, Tipu last_name: Sultan - first_name: Mehran Beiraghi full_name: Toosi, Mehran Beiraghi last_name: Toosi - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 - first_name: Mariasavina full_name: Severino, Mariasavina last_name: Severino - first_name: Henry full_name: Houlden, Henry last_name: Houlden - first_name: Robert W. full_name: Taylor, Robert W. last_name: Taylor - first_name: Reza full_name: Maroofian, Reza last_name: Maroofian citation: ama: Kaiyrzhanov R, Thompson K, Efthymiou S, et al. Biallelic NDUFA13 variants lead to a neurodevelopmental phenotype with gradual neurological impairment. Brain Communications. 2025;7(1). doi:10.1093/braincomms/fcae453 apa: Kaiyrzhanov, R., Thompson, K., Efthymiou, S., Mukushev, A., Zharylkassyn, A., Prasad, C., … Maroofian, R. (2025). Biallelic NDUFA13 variants lead to a neurodevelopmental phenotype with gradual neurological impairment. Brain Communications. Oxford University Press. https://doi.org/10.1093/braincomms/fcae453 chicago: Kaiyrzhanov, Rauan, Kyle Thompson, Stephanie Efthymiou, Askhat Mukushev, Akbota Zharylkassyn, Chitra Prasad, Ehsan Ghayoor Karimiani, et al. “Biallelic NDUFA13 Variants Lead to a Neurodevelopmental Phenotype with Gradual Neurological Impairment.” Brain Communications. Oxford University Press, 2025. https://doi.org/10.1093/braincomms/fcae453. ieee: R. Kaiyrzhanov et al., “Biallelic NDUFA13 variants lead to a neurodevelopmental phenotype with gradual neurological impairment,” Brain Communications, vol. 7, no. 1. Oxford University Press, 2025. ista: Kaiyrzhanov R, Thompson K, Efthymiou S, Mukushev A, Zharylkassyn A, Prasad C, Karimiani EG, Alvi JR, Niyazov D, Alahmad A, Babaei M, Tajsharghi H, Albash B, Alaqeel A, Charif M, Hashemi N, Heidari M, Kalantar SM, Lenaers G, Mehrjardi MYV, Srinivasan VM, Gowda VK, Mirabutalebi SH, Carere DA, Movahedinia M, Murphy D, Mcfarland R, Abdel-Hamid MS, Elhossini RM, Alavi S, Napier M, Belanger-Quintana A, Prasad AN, Jakobczyk J, Roubertie A, Rupar T, Sultan T, Toosi MB, Sazanov LA, Severino M, Houlden H, Taylor RW, Maroofian R. 2025. Biallelic NDUFA13 variants lead to a neurodevelopmental phenotype with gradual neurological impairment. Brain Communications. 7(1), fcae453. mla: Kaiyrzhanov, Rauan, et al. “Biallelic NDUFA13 Variants Lead to a Neurodevelopmental Phenotype with Gradual Neurological Impairment.” Brain Communications, vol. 7, no. 1, fcae453, Oxford University Press, 2025, doi:10.1093/braincomms/fcae453. short: R. Kaiyrzhanov, K. Thompson, S. Efthymiou, A. Mukushev, A. Zharylkassyn, C. Prasad, E.G. Karimiani, J.R. Alvi, D. Niyazov, A. Alahmad, M. Babaei, H. Tajsharghi, B. Albash, A. Alaqeel, M. Charif, N. Hashemi, M. Heidari, S.M. Kalantar, G. Lenaers, M.Y.V. Mehrjardi, V.M. Srinivasan, V.K. Gowda, S.H. Mirabutalebi, D.A. Carere, M. Movahedinia, D. Murphy, R. Mcfarland, M.S. Abdel-Hamid, R.M. Elhossini, S. Alavi, M. Napier, A. Belanger-Quintana, A.N. Prasad, J. Jakobczyk, A. Roubertie, T. Rupar, T. Sultan, M.B. Toosi, L.A. Sazanov, M. Severino, H. Houlden, R.W. Taylor, R. Maroofian, Brain Communications 7 (2025). date_created: 2025-02-02T23:01:55Z date_published: 2025-01-01T00:00:00Z date_updated: 2025-08-05T11:55:15Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1093/braincomms/fcae453 external_id: pmid: - '39963288' file: - access_level: open_access checksum: bdf39b64d1c1d833a20b62836751fe44 content_type: application/pdf creator: dernst date_created: 2025-08-05T11:54:23Z date_updated: 2025-08-05T11:54:23Z file_id: '20126' file_name: 2025_BrainComm_Kaiyrzhanov.pdf file_size: 1420646 relation: main_file success: 1 file_date_updated: 2025-08-05T11:54:23Z has_accepted_license: '1' intvolume: ' 7' issue: '1' language: - iso: eng month: '01' oa: 1 oa_version: Published Version pmid: 1 publication: Brain Communications publication_identifier: eissn: - 2632-1297 publication_status: published publisher: Oxford University Press quality_controlled: '1' scopus_import: '1' status: public title: Biallelic NDUFA13 variants lead to a neurodevelopmental phenotype with gradual neurological impairment tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 7 year: '2025' ... --- DOAJ_listed: '1' OA_place: publisher OA_type: gold _id: '19529' abstract: - lang: eng text: NRF2 is a transcription factor responsible for coordinating the expression of over a thousand cytoprotective genes. Although NRF2 is constitutively expressed, its stability is modulated by the redox-sensitive protein KEAP1 and other conditional binding partner regulators. The new era of NRF2 research has highlighted the cooperation between NRF2 and PIN1 in modifying its cytoprotective effect. Despite numerous studies, the understanding of the PIN1-NRF2 interaction remains limited. Herein, we described the binding interaction of PIN1 and three different 14-mer long phospho-peptides mimicking NRF2 protein using computer-based, biophysical, and biochemical approaches. According to our computational analyses, the residues positioned in the WW domain of PIN1 (Ser16, Arg17, Ser18, Tyr23, Ser32, Gln33, and Trp34) were found to be crucial for PIN1-NRF2 interactions. Biophysical FP assays were used to verify the computational prediction. The data demonstrated that Pintide, a peptide predominantly interacting with the PIN1 WW-domain, led to a significant reduction in the binding affinity of the NRF2 mimicking peptides. Moreover, we evaluated the impact of known PIN1 inhibitors (juglone, KPT-6566, and EGCG) on the PIN1-NRF2 interaction. Among the inhibitors, KPT-6566 showed the most potent inhibitory effect on PIN1-NRF2 interaction within an IC50 range of 0.3–1.4 µM. Furthermore, our mass spectrometry analyses showed that KPT-6566 appeared to covalently modify PIN1 via conjugate addition, rather than disulfide exchange of the sulfonyl-acetate moiety. Altogether, such inhibitors would also be highly valuable molecular probes for further investigation of PIN1 regulation of NRF2 in the cellular context and potentially pave the way for drug molecules that specifically inhibit the cytoprotective effects of NRF2 in cancer. acknowledgement: The authors would like to thank the Ministry of National Education of Republic of Türkiye within the scope of the YLSY scholarship program for funding (AO). This article is based upon work from COST Action CA20121, supported by COST (European Cooperation in Science and Technology) (www.cost.eu) (https://benbedphar.org/about-benbedphar/). The molecular dynamics simulations reported in this paper were performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). The authors thank Dr Sharad Mistry for his support in acquiring and processing the MS data. article_number: '8907' article_processing_charge: Yes article_type: original author: - first_name: Adem full_name: Ozleyen, Adem last_name: Ozleyen - first_name: Gizem Nur full_name: Duran, Gizem Nur last_name: Duran - first_name: Serhat full_name: Dönmez, Serhat id: 7c624079-3200-11ee-973b-9fcc8a575580 last_name: Dönmez - first_name: Mehmet full_name: Ozbil, Mehmet last_name: Ozbil - first_name: Richard G. full_name: Doveston, Richard G. last_name: Doveston - first_name: Tugba Boyunegmez full_name: Tumer, Tugba Boyunegmez last_name: Tumer citation: ama: Ozleyen A, Duran GN, Dönmez S, Ozbil M, Doveston RG, Tumer TB. Identification and inhibition of PIN1-NRF2 protein–protein interactions through computational and biophysical approaches. Scientific Reports. 2025;15. doi:10.1038/s41598-025-89342-0 apa: Ozleyen, A., Duran, G. N., Dönmez, S., Ozbil, M., Doveston, R. G., & Tumer, T. B. (2025). Identification and inhibition of PIN1-NRF2 protein–protein interactions through computational and biophysical approaches. Scientific Reports. Springer Nature. https://doi.org/10.1038/s41598-025-89342-0 chicago: Ozleyen, Adem, Gizem Nur Duran, Serhat Dönmez, Mehmet Ozbil, Richard G. Doveston, and Tugba Boyunegmez Tumer. “Identification and Inhibition of PIN1-NRF2 Protein–Protein Interactions through Computational and Biophysical Approaches.” Scientific Reports. Springer Nature, 2025. https://doi.org/10.1038/s41598-025-89342-0. ieee: A. Ozleyen, G. N. Duran, S. Dönmez, M. Ozbil, R. G. Doveston, and T. B. Tumer, “Identification and inhibition of PIN1-NRF2 protein–protein interactions through computational and biophysical approaches,” Scientific Reports, vol. 15. Springer Nature, 2025. ista: Ozleyen A, Duran GN, Dönmez S, Ozbil M, Doveston RG, Tumer TB. 2025. Identification and inhibition of PIN1-NRF2 protein–protein interactions through computational and biophysical approaches. Scientific Reports. 15, 8907. mla: Ozleyen, Adem, et al. “Identification and Inhibition of PIN1-NRF2 Protein–Protein Interactions through Computational and Biophysical Approaches.” Scientific Reports, vol. 15, 8907, Springer Nature, 2025, doi:10.1038/s41598-025-89342-0. short: A. Ozleyen, G.N. Duran, S. Dönmez, M. Ozbil, R.G. Doveston, T.B. Tumer, Scientific Reports 15 (2025). date_created: 2025-04-08T11:12:20Z date_published: 2025-03-14T00:00:00Z date_updated: 2025-09-30T11:33:37Z day: '14' ddc: - '570' department: - _id: LeSa doi: 10.1038/s41598-025-89342-0 external_id: isi: - '001445507400002' pmid: - '40087364' file: - access_level: open_access checksum: 6124a10402a67b66364cfa9350d35b4b content_type: application/pdf creator: dernst date_created: 2025-04-10T06:21:11Z date_updated: 2025-04-10T06:21:11Z file_id: '19537' file_name: 2025_ScientificReports_Ozleyen.pdf file_size: 5333058 relation: main_file success: 1 file_date_updated: 2025-04-10T06:21:11Z has_accepted_license: '1' intvolume: ' 15' isi: 1 language: - iso: eng month: '03' oa: 1 oa_version: Published Version pmid: 1 publication: Scientific Reports publication_identifier: eissn: - 2045-2322 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Identification and inhibition of PIN1-NRF2 protein–protein interactions through computational and biophysical approaches tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345 volume: 15 year: '2025' ... --- DOAJ_listed: '1' OA_place: publisher OA_type: gold PlanS_conform: '1' _id: '20002' abstract: - lang: eng text: While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts. acknowledgement: We thank Dr. Kenneth Johnson for assistance with kinetic analysis and helpful discussion as well as Dr. Jack Bravo and members of the Taylor lab for insightful comments on the manuscript. Data were collected at the Sauer Structural Biology Laboratory at the University of Texas at Austin. This work was supported by a National Institutes of Health grant R35GM138348 (to D.W.T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Computational resources for this work were supported by the Welch Foundation grant F-1938 (to D.W.T.). article_number: '5681' article_processing_charge: Yes article_type: original author: - first_name: Kaitlyn full_name: Kiernan, Kaitlyn id: 91e8ab53-b70a-11ef-adcb-f779f833b451 last_name: Kiernan - first_name: David W. full_name: Taylor, David W. last_name: Taylor citation: ama: Kiernan K, Taylor DW. Visualization of a multi-turnover Cas9 after product release. Nature Communications. 2025;16. doi:10.1038/s41467-025-60668-7 apa: Kiernan, K., & Taylor, D. W. (2025). Visualization of a multi-turnover Cas9 after product release. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-025-60668-7 chicago: Kiernan, Kaitlyn, and David W. Taylor. “Visualization of a Multi-Turnover Cas9 after Product Release.” Nature Communications. Springer Nature, 2025. https://doi.org/10.1038/s41467-025-60668-7. ieee: K. Kiernan and D. W. Taylor, “Visualization of a multi-turnover Cas9 after product release,” Nature Communications, vol. 16. Springer Nature, 2025. ista: Kiernan K, Taylor DW. 2025. Visualization of a multi-turnover Cas9 after product release. Nature Communications. 16, 5681. mla: Kiernan, Kaitlyn, and David W. Taylor. “Visualization of a Multi-Turnover Cas9 after Product Release.” Nature Communications, vol. 16, 5681, Springer Nature, 2025, doi:10.1038/s41467-025-60668-7. short: K. Kiernan, D.W. Taylor, Nature Communications 16 (2025). date_created: 2025-07-13T22:01:21Z date_published: 2025-07-01T00:00:00Z date_updated: 2025-07-14T08:30:06Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1038/s41467-025-60668-7 external_id: pmid: - '40593576' file: - access_level: open_access checksum: fa9a1eaa7e2e60467768cbaed307aceb content_type: application/pdf creator: dernst date_created: 2025-07-14T08:28:25Z date_updated: 2025-07-14T08:28:25Z file_id: '20018' file_name: 2025_NatureComm_Kiernan.pdf file_size: 6875712 relation: main_file success: 1 file_date_updated: 2025-07-14T08:28:25Z has_accepted_license: '1' intvolume: ' 16' language: - iso: eng month: '07' oa: 1 oa_version: Published Version pmid: 1 publication: Nature Communications publication_identifier: eissn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Visualization of a multi-turnover Cas9 after product release tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 16 year: '2025' ... --- APC_amount: 12348 EUR OA_place: publisher OA_type: hybrid _id: '17884' abstract: - lang: eng text: Human T cell leukemia virus type 1 (HTLV-1) immature particles differ in morphology from other retroviruses, suggesting a distinct way of assembly. Here we report the results of cryo-electron tomography studies of HTLV-1 virus-like particles assembled in vitro, as well as derived from cells. This work shows that HTLV-1 uses a distinct mechanism of Gag–Gag interactions to form the immature viral lattice. Analysis of high-resolution structural information from immature capsid (CA) tubular arrays reveals that the primary stabilizing component in HTLV-1 is the N-terminal domain of CA. Mutagenesis analysis supports this observation. This distinguishes HTLV-1 from other retroviruses, in which the stabilization is provided primarily by the C-terminal domain of CA. These results provide structural details of the quaternary arrangement of Gag for an immature deltaretrovirus and this helps explain why HTLV-1 particles are morphologically distinct. acknowledged_ssus: - _id: ScienComp - _id: LifeSc - _id: EM-Fac acknowledgement: This work was funded by the Institute of Science and Technology Austria (ISTA) and the Austrian Science Fund (grant P31445 to F.K.M.S.). Access to high-resolution cryo-ET data acquisition at European Molecular Biology Laboratory (EMBL) Heidelberg was supported through the EMBL cryo-EM platform. We thank V.-V. Hodirnau at ISTA and W. Hagen and F. Weis at EMBL Heidelberg for support in cryo-ET data acquisition. This research was also supported by the scientific service units of ISTA through resources provided by Scientific Computing, the Life Science Facility, and the EM Facility. L.M.M. was supported by National Institutes of Health grants R01 GM151775 and R21 DE032878 and by the University of Minnesota Masonic Cancer Center. D.P. was supported by the DOC doctoral fellowship program of the Austrian Academy of Sciences. R.A.D was supported by the National Institute of Allergy and Infectious Diseases (grant R01AI147890). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Specifically, we also want to thank A. Schlögl for computational support and J. Hansen and V. Vogt for critical comments on the manuscript. We also thank the other members of the Schur lab for helpful discussions and experimental advice. article_processing_charge: Yes (in subscription journal) article_type: original author: - first_name: Martin full_name: Obr, Martin id: 4741CA5A-F248-11E8-B48F-1D18A9856A87 last_name: Obr orcid: 0000-0003-1756-6564 - first_name: Mathias full_name: Percipalle, Mathias id: 4986e21c-eb97-11eb-a6c2-a4ef0b629971 last_name: Percipalle - first_name: Darya full_name: Chernikova, Darya id: 7dbaf460-fa9e-11eb-b0ca-bc7c7ff21ad0 last_name: Chernikova - first_name: Huixin full_name: Yang, Huixin last_name: Yang - first_name: Andreas full_name: Thader, Andreas id: 3A18A7B8-F248-11E8-B48F-1D18A9856A87 last_name: Thader - first_name: Gergely full_name: Pinke, Gergely id: 4D5303E6-F248-11E8-B48F-1D18A9856A87 last_name: Pinke - first_name: Dario J full_name: Porley, Dario J id: 2FD6EA6C-F248-11E8-B48F-1D18A9856A87 last_name: Porley - first_name: Louis M. full_name: Mansky, Louis M. last_name: Mansky - first_name: Robert A. full_name: Dick, Robert A. last_name: Dick - first_name: Florian KM full_name: Schur, Florian KM id: 48AD8942-F248-11E8-B48F-1D18A9856A87 last_name: Schur orcid: 0000-0003-4790-8078 citation: ama: Obr M, Percipalle M, Chernikova D, et al. Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice. Nature Structural & Molecular Biology. 2025;32:268-276. doi:10.1038/s41594-024-01390-8 apa: Obr, M., Percipalle, M., Chernikova, D., Yang, H., Thader, A., Pinke, G., … Schur, F. K. (2025). Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice. Nature Structural & Molecular Biology. Springer Nature. https://doi.org/10.1038/s41594-024-01390-8 chicago: Obr, Martin, Mathias Percipalle, Darya Chernikova, Huixin Yang, Andreas Thader, Gergely Pinke, Darío Porley Esteves, Louis M. Mansky, Robert A. Dick, and Florian KM Schur. “Distinct Stabilization of the Human T Cell Leukemia Virus Type 1 Immature Gag Lattice.” Nature Structural & Molecular Biology. Springer Nature, 2025. https://doi.org/10.1038/s41594-024-01390-8. ieee: M. Obr et al., “Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice,” Nature Structural & Molecular Biology, vol. 32. Springer Nature, pp. 268–276, 2025. ista: Obr M, Percipalle M, Chernikova D, Yang H, Thader A, Pinke G, Porley Esteves D, Mansky LM, Dick RA, Schur FK. 2025. Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice. Nature Structural & Molecular Biology. 32, 268–276. mla: Obr, Martin, et al. “Distinct Stabilization of the Human T Cell Leukemia Virus Type 1 Immature Gag Lattice.” Nature Structural & Molecular Biology, vol. 32, Springer Nature, 2025, pp. 268–76, doi:10.1038/s41594-024-01390-8. short: M. Obr, M. Percipalle, D. Chernikova, H. Yang, A. Thader, G. Pinke, D. Porley Esteves, L.M. Mansky, R.A. Dick, F.K. Schur, Nature Structural & Molecular Biology 32 (2025) 268–276. corr_author: '1' date_created: 2024-09-08T10:29:06Z date_published: 2025-02-01T00:00:00Z date_updated: 2026-03-16T12:55:18Z day: '01' ddc: - '570' department: - _id: FlSc - _id: LeSa doi: 10.1038/s41594-024-01390-8 external_id: isi: - '001306564000001' oaworkid: - W4402316284 pmid: - '39242978' file: - access_level: open_access checksum: c641ad94afb28917b20425db676fc3ee content_type: application/pdf creator: dernst date_created: 2025-04-23T07:02:33Z date_updated: 2025-04-23T07:02:33Z file_id: '19608' file_name: 2025_NatureStrucBio_Obr.pdf file_size: 13724041 relation: main_file success: 1 file_date_updated: 2025-04-23T07:02:33Z has_accepted_license: '1' intvolume: ' 32' isi: 1 language: - iso: eng month: '02' oa: 1 oa_version: Published Version oaworkid: 1 page: 268-276 pmid: 1 project: - _id: 26736D6A-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: P31445 name: Structural conservation and diversity in retroviral capsid - _id: 9B9C98E0-BA93-11EA-9121-9846C619BF3A grant_number: '25762' name: Structural characterization of spumavirus capsid assemblies to understand conserved Ortervirales assembly mechanisms publication: Nature Structural & Molecular Biology publication_identifier: eissn: - 1545-9985 issn: - 1545-9993 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 32 year: '2025' ... --- OA_place: publisher OA_type: hybrid _id: '18170' abstract: - lang: eng text: 'This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.' acknowledgement: 'This research was funded in whole by the Austrian Science Fund (FWF) [P 35103-B, Grant-DOI: 10.55776/P35103]. For open access purposes, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission. We would like to thank Olfert Landt for advice on ssDNA probe design; Rui Qiang Chen, Jennifer Stock, and Christine Wukotitsch for their excellent support with ONT sequencing; Christoph Köppl and Andreas Fischer for excellent support in recombinant N protein expression and purification; and the whole team at the division of clinical virology for their support with standard diagnostics.' article_number: '116807' article_processing_charge: Yes (in subscription journal) article_type: original author: - first_name: Anna Nele full_name: Herdina, Anna Nele last_name: Herdina - first_name: Anil full_name: Bozdogan, Anil last_name: Bozdogan - first_name: Patrik full_name: Aspermair, Patrik last_name: Aspermair - first_name: Jakub full_name: Dostalek, Jakub last_name: Dostalek - first_name: Miriam full_name: Klausberger, Miriam last_name: Klausberger - first_name: Nico full_name: Lingg, Nico last_name: Lingg - first_name: Monika full_name: Cserjan-Puschmann, Monika last_name: Cserjan-Puschmann - first_name: Patricia Pereira full_name: Aguilar, Patricia Pereira last_name: Aguilar - first_name: Simone full_name: Auer, Simone last_name: Auer - first_name: Halil full_name: Demirtas, Halil last_name: Demirtas - first_name: Jakob full_name: Andersson, Jakob id: 3a5f4167-9bd9-11ed-bd12-a1446d38776f last_name: Andersson - first_name: Felix full_name: Lötsch, Felix last_name: Lötsch - first_name: Barbara full_name: Holzer, Barbara last_name: Holzer - first_name: Adi full_name: Steinrigl, Adi last_name: Steinrigl - first_name: Florian full_name: Thalhammer, Florian last_name: Thalhammer - first_name: Julia full_name: Schellnegger, Julia last_name: Schellnegger - first_name: Monika full_name: Breuer, Monika last_name: Breuer - first_name: Wolfgang full_name: Knoll, Wolfgang last_name: Knoll - first_name: Robert full_name: Strassl, Robert last_name: Strassl citation: ama: 'Herdina AN, Bozdogan A, Aspermair P, et al. Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements. Biosensors and Bioelectronics. 2025;267. doi:10.1016/j.bios.2024.116807' apa: 'Herdina, A. N., Bozdogan, A., Aspermair, P., Dostalek, J., Klausberger, M., Lingg, N., … Strassl, R. (2025). Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements. Biosensors and Bioelectronics. Elsevier. https://doi.org/10.1016/j.bios.2024.116807' chicago: 'Herdina, Anna Nele, Anil Bozdogan, Patrik Aspermair, Jakub Dostalek, Miriam Klausberger, Nico Lingg, Monika Cserjan-Puschmann, et al. “Bridging Basic Science and Applied Diagnostics: Comprehensive Viral Diagnostics Enabled by Graphene-Based Electronic Biosensor Technology Advancements.” Biosensors and Bioelectronics. Elsevier, 2025. https://doi.org/10.1016/j.bios.2024.116807.' ieee: 'A. N. Herdina et al., “Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements,” Biosensors and Bioelectronics, vol. 267. Elsevier, 2025.' ista: 'Herdina AN, Bozdogan A, Aspermair P, Dostalek J, Klausberger M, Lingg N, Cserjan-Puschmann M, Aguilar PP, Auer S, Demirtas H, Andersson J, Lötsch F, Holzer B, Steinrigl A, Thalhammer F, Schellnegger J, Breuer M, Knoll W, Strassl R. 2025. Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements. Biosensors and Bioelectronics. 267, 116807.' mla: 'Herdina, Anna Nele, et al. “Bridging Basic Science and Applied Diagnostics: Comprehensive Viral Diagnostics Enabled by Graphene-Based Electronic Biosensor Technology Advancements.” Biosensors and Bioelectronics, vol. 267, 116807, Elsevier, 2025, doi:10.1016/j.bios.2024.116807.' short: A.N. Herdina, A. Bozdogan, P. Aspermair, J. Dostalek, M. Klausberger, N. Lingg, M. Cserjan-Puschmann, P.P. Aguilar, S. Auer, H. Demirtas, J. Andersson, F. Lötsch, B. Holzer, A. Steinrigl, F. Thalhammer, J. Schellnegger, M. Breuer, W. Knoll, R. Strassl, Biosensors and Bioelectronics 267 (2025). date_created: 2024-10-06T22:01:11Z date_published: 2025-01-01T00:00:00Z date_updated: 2025-02-27T12:34:07Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1016/j.bios.2024.116807 external_id: isi: - '001328413700001' pmid: - '39341071' file: - access_level: open_access checksum: 208ac27dab27af792d198fcb74af8756 content_type: application/pdf creator: dernst date_created: 2025-01-13T11:14:32Z date_updated: 2025-01-13T11:14:32Z file_id: '18843' file_name: 2025_BiosensorsBioelectronics_Herdina.pdf file_size: 4135372 relation: main_file success: 1 file_date_updated: 2025-01-13T11:14:32Z has_accepted_license: '1' intvolume: ' 267' isi: 1 language: - iso: eng month: '01' oa: 1 oa_version: Published Version pmid: 1 publication: Biosensors and Bioelectronics publication_identifier: eissn: - 1873-4235 issn: - 0956-5663 publication_status: published publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: 'Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements' tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 267 year: '2025' ... --- OA_place: publisher OA_type: hybrid _id: '18967' abstract: - lang: eng text: "Background: We identified small molecule tricyclic pyrone compound CP2 as a mild mitochondrial complex I (MCI) inhibitor that induces neuroprotection in multiple mouse models of AD. One of the major concerns while targeting mitochondria is the production of reactive oxygen species (ROS). CP2 consists of two diastereoisomers, D1 and D2, with distinct activity and toxicity profiles. This study was designed to understand how structure of D1 and D2 affects their binding to MCI and the consequential impact on ROS production.\r\n\r\nMethod: The X-ray crystallography and cryo-electron microscopy (cryo-EM) at global resolution of 3.25-3.27Å were employed to identify the molecular structure of D1 and D2 and the D1 binding to the isolated ovine MCI. The assessment of the MCI inhibition and the extent of ROS generation were done in isolated MCI and human neuroblastoma MC65 cells using flow cytometry, a Seahorse extracellular flux analyzer, and the kinetic studies.\r\n\r\nResult: In the closed conformation of MCI, D1 selectively binds to the deep Quinone-site (Qd) but not to the shallow Q-site (Qs), sharing the same binding pocket as rotenone. In the open MCI state, D1 exclusively binds to the Qs in contrast to rotenone, which binds Qd and Qs in both closed and open states. At the same concentrations, D1 inhibits respiration to a greater extent compared to D2 (5:1 ratio) and produces higher level of ROS.\r\n\r\nConclusion:Cryo-EM unambiguously identified binding of D1 to both the Qd and Qs sites, contingent upon the conformational state of MCI. In contrast to rotenone, D1 binds Qd only in the closed conformation during catalytic cycle, leading to mild inhibition. Superimposing X-ray crystallography data of D1 and D2 onto cryo-EM data suggests that the orientation of the methyl group in D2 induces a flatter conformation, resulting in lower binding affinity to MCI, which correlates with lower inhibition and toxicity compared to D1. At physiologically relevant concentrations, CP2 (D1:D2 = 1:1) demonstrates low MCI inhibition yielding negligible ROS levels. This observation provides new insight into the absence of toxicity associated with CP2 treatment in vivo, further highlighting feasibility for the development of safe and efficacious MCI inhibitors." article_number: e085971 article_processing_charge: Yes (in subscription journal) author: - first_name: Olga full_name: Petrova, Olga id: 5D8C9660-5D49-11EA-8188-567B3DDC885E last_name: Petrova - first_name: Sergey A full_name: Trushin, Sergey A last_name: Trushin - first_name: Thi Kim Oanh full_name: Nguyen, Thi Kim Oanh last_name: Nguyen - first_name: Mark full_name: Ostroot, Mark last_name: Ostroot - first_name: Matthew full_name: Schellenberg, Matthew last_name: Schellenberg - first_name: Graham full_name: Johnson, Graham last_name: Johnson - first_name: Eugenia full_name: Trushina, Eugenia last_name: Trushina - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Petrova O, Trushin SA, Nguyen TKO, et al. Structure‐activity Relationship Study of Neuroprotective Complex I Inhibitor CP2. Vol 20. Wiley; 2024. doi:10.1002/alz.085971 apa: Petrova, O., Trushin, S. A., Nguyen, T. K. O., Ostroot, M., Schellenberg, M., Johnson, G., … Sazanov, L. A. (2024). Structure‐activity relationship study of neuroprotective complex I inhibitor CP2. Alzheimer’s & Dementia (Vol. 20). Wiley. https://doi.org/10.1002/alz.085971 chicago: Petrova, Olga, Sergey A Trushin, Thi Kim Oanh Nguyen, Mark Ostroot, Matthew Schellenberg, Graham Johnson, Eugenia Trushina, and Leonid A Sazanov. Structure‐activity Relationship Study of Neuroprotective Complex I Inhibitor CP2. Alzheimer’s & Dementia. Vol. 20. Wiley, 2024. https://doi.org/10.1002/alz.085971. ieee: O. Petrova et al., Structure‐activity relationship study of neuroprotective complex I inhibitor CP2, vol. 20, no. S6. Wiley, 2024. ista: Petrova O, Trushin SA, Nguyen TKO, Ostroot M, Schellenberg M, Johnson G, Trushina E, Sazanov LA. 2024. Structure‐activity relationship study of neuroprotective complex I inhibitor CP2, Wiley,p. mla: Petrova, Olga, et al. “Structure‐activity Relationship Study of Neuroprotective Complex I Inhibitor CP2.” Alzheimer’s & Dementia, vol. 20, no. S6, e085971, Wiley, 2024, doi:10.1002/alz.085971. short: O. Petrova, S.A. Trushin, T.K.O. Nguyen, M. Ostroot, M. Schellenberg, G. Johnson, E. Trushina, L.A. Sazanov, Structure‐activity Relationship Study of Neuroprotective Complex I Inhibitor CP2, Wiley, 2024. date_created: 2025-01-29T15:21:40Z date_published: 2024-12-01T00:00:00Z date_updated: 2025-01-29T15:29:22Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1002/alz.085971 file: - access_level: open_access checksum: e914bd5f3a701659ab79d497a122811f content_type: application/pdf creator: dernst date_created: 2025-01-29T15:24:50Z date_updated: 2025-01-29T15:24:50Z file_id: '18968' file_name: 2024_AlzheimerDementia_Petrova.pdf file_size: 70870 relation: main_file success: 1 file_date_updated: 2025-01-29T15:24:50Z has_accepted_license: '1' intvolume: ' 20' issue: S6 language: - iso: eng month: '12' oa: 1 oa_version: Published Version publication: Alzheimer's & Dementia publication_identifier: eissn: - 1552-5279 issn: - 1552-5260 publication_status: published publisher: Wiley quality_controlled: '1' status: public title: Structure‐activity relationship study of neuroprotective complex I inhibitor CP2 tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: other_academic_publication user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 20 year: '2024' ... --- _id: '15323' abstract: - lang: eng text: Supercomplexes of the respiratory chain are established constituents of the oxidative phosphorylation system, but their role in mammalian metabolism has been hotly debated. Although recent studies have shown that different tissues/organs are equipped with specific sets of supercomplexes, depending on their metabolic needs, the notion that supercomplexes have a role in the regulation of metabolism has been challenged. However, irrespective of the mechanistic conclusions, the composition of various high molecular weight supercomplexes remains uncertain. Here, using cryogenic electron microscopy, we demonstrate that mammalian (mouse) tissues contain three defined types of ‘respirasome’, supercomplexes made of CI, CIII2 and CIV. The stoichiometry and position of CIV differs in the three respirasomes, of which only one contains the supercomplex-associated factor SCAF1, whose involvement in respirasome formation has long been contended. Our structures confirm that the ‘canonical’ respirasome (the C-respirasome, CICIII2CIV) does not contain SCAF1, which is instead associated to a different respirasome (the CS-respirasome), containing a second copy of CIV. We also identify an alternative respirasome (A-respirasome), with CIV bound to the ‘back’ of CI, instead of the ‘toe’. This structural characterization of mouse mitochondrial supercomplexes allows us to hypothesize a mechanistic basis for their specific role in different metabolic conditions. acknowledged_ssus: - _id: EM-Fac - _id: LifeSc - _id: PreCl - _id: ScienComp acknowledgement: Supercomplexes of the respiratory chain are established constituents of the oxidative phosphorylation system, but their role in mammalian metabolism has been hotly debated. Although recent studies have shown that different tissues/organs are equipped with specific sets of supercomplexes, depending on their metabolic needs, the notion that supercomplexes have a role in the regulation of metabolism has been challenged. However, irrespective of the mechanistic conclusions, the composition of various high molecular weight supercomplexes remains uncertain. Here, using cryogenic electron microscopy, we demonstrate that mammalian (mouse) tissues contain three defined types of ‘respirasome’, supercomplexes made of CI, CIII2 and CIV. The stoichiometry and position of CIV differs in the three respirasomes, of which only one contains the supercomplex-associated factor SCAF1, whose involvement in respirasome formation has long been contended. Our structures confirm that the ‘canonical’ respirasome (the C-respirasome, CICIII2CIV) does not contain SCAF1, which is instead associated to a different respirasome (the CS-respirasome), containing a second copy of CIV. We also identify an alternative respirasome (A-respirasome), with CIV bound to the ‘back’ of CI, instead of the ‘toe’. This structural characterization of mouse mitochondrial supercomplexes allows us to hypothesize a mechanistic basis for their specific role in different metabolic conditions. article_processing_charge: No article_type: original author: - first_name: Irene full_name: Vercellino, Irene id: 3ED6AF16-F248-11E8-B48F-1D18A9856A87 last_name: Vercellino orcid: 0000-0001-5618-3449 - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Vercellino I, Sazanov LA. SCAF1 drives the compositional diversity of mammalian respirasomes. Nature Structural and Molecular Biology. 2024;31:1061-1071. doi:10.1038/s41594-024-01255-0 apa: Vercellino, I., & Sazanov, L. A. (2024). SCAF1 drives the compositional diversity of mammalian respirasomes. Nature Structural and Molecular Biology. Springer Nature. https://doi.org/10.1038/s41594-024-01255-0 chicago: Vercellino, Irene, and Leonid A Sazanov. “SCAF1 Drives the Compositional Diversity of Mammalian Respirasomes.” Nature Structural and Molecular Biology. Springer Nature, 2024. https://doi.org/10.1038/s41594-024-01255-0. ieee: I. Vercellino and L. A. Sazanov, “SCAF1 drives the compositional diversity of mammalian respirasomes,” Nature Structural and Molecular Biology, vol. 31. Springer Nature, pp. 1061–1071, 2024. ista: Vercellino I, Sazanov LA. 2024. SCAF1 drives the compositional diversity of mammalian respirasomes. Nature Structural and Molecular Biology. 31, 1061–1071. mla: Vercellino, Irene, and Leonid A. Sazanov. “SCAF1 Drives the Compositional Diversity of Mammalian Respirasomes.” Nature Structural and Molecular Biology, vol. 31, Springer Nature, 2024, pp. 1061–71, doi:10.1038/s41594-024-01255-0. short: I. Vercellino, L.A. Sazanov, Nature Structural and Molecular Biology 31 (2024) 1061–1071. corr_author: '1' date_created: 2024-04-14T22:01:03Z date_published: 2024-07-01T00:00:00Z date_updated: 2025-11-24T08:35:04Z day: '01' ddc: - '572' department: - _id: LeSa doi: 10.1038/s41594-024-01255-0 ec_funded: 1 external_id: isi: - '001196897300001' pmid: - '38575788' file: - access_level: open_access checksum: 21f05d188762acd7f49a97f3d09c8d9f content_type: application/pdf creator: lsazanov date_created: 2024-05-14T11:57:56Z date_updated: 2025-01-01T23:30:03Z embargo: 2025-01-01 file_id: '15392' file_name: megacomplex_submit_NSMB_withFigures.pdf file_size: 24424729 relation: main_file file_date_updated: 2025-01-01T23:30:03Z has_accepted_license: '1' intvolume: ' 31' isi: 1 language: - iso: eng month: '07' oa: 1 oa_version: Submitted Version page: 1061-1071 pmid: 1 project: - _id: 627abdeb-2b32-11ec-9570-ec31a97243d3 call_identifier: H2020 grant_number: '101020697' name: Structure and mechanism of respiratory chain molecular machines publication: Nature Structural and Molecular Biology publication_identifier: eissn: - 1545-9985 issn: - 1545-9993 publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: link: - relation: erratum url: https://doi.org/10.1038/s41594-025-01721-3 scopus_import: '1' status: public title: SCAF1 drives the compositional diversity of mammalian respirasomes tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 31 year: '2024' ... --- OA_place: publisher _id: '17319' abstract: - lang: eng text: "This thesis comprises two distinct projects, each offering unique insights into fundamental\r\ncellular processes. While distinct in their focus, these different perspectives have a common\r\ntheme: chemiosmotic theory and utilisation of the proton gradient for driving the essential\r\nprocesses like auxin efflux and ATP synthesis, effectively bridging the membrane protein\r\nstructure and function from the realms of plant biology and cellular bioenergetics.\r\nThe first project of this thesis centres on the characterisation of PIN proteins, a class of\r\ntransmembrane transporters pivotal in the regulation of auxin transport and distribution in\r\nplants. PINs form a conserved and phylogenetically abundant group of transporters present in\r\nland plants and certain algae. Despite their great importance, they were one of the few elusive\r\nproteins essential for plant development not to be structurally and mechanistically\r\ncharacterised since their discovery almost 30 years ago. This work aimed to uncover the\r\nstructural and functional dynamics of the PIN protein-mediated auxin transport using an array\r\nof experimental techniques, including protein purification, biochemical assays and structural\r\nanalysis. Through an exhaustive screening process that took several years and included testing\r\ndifferent PIN homologues, expression systems, constructs, and purification conditions, we\r\ndeveloped a robust protocol for isolating the pure, stable, and monodisperse PIN8 protein.\r\nMoreover, utilising biophysical methods and buffer screening, we demonstrated that PIN8\r\nexhibits detergent and pH-dependent stability, with mild detergents and lower pH (5.0 and 6.0)\r\nbeing optimal for the stability of the protein. Using SEC-MALS and crosslinking, we\r\ndetermined that PIN8 forms dimers, which was confirmed by our structural studies. We\r\nobtained a cryo-EM map of PIN8 at pH 6.0, and, compared to recently published structures,\r\nour map implies major pH-dependent conformational changes and possibly utilisation of the\r\nproton gradient in the transport mechanism.\r\nThe subject of the second project was F1Fo-ATP synthase, an enzyme complex fundamental\r\nto cellular energy metabolism. Through an approach integrating biochemical assays and\r\nstructural analysis, this research aimed to unveil the molecular mechanism of inhibition of ATP\r\nsynthase by yaku´amide, a bioactive compound with potential therapeutic implications. Using\r\nsubmitochondrial particles and purified F1Fo-ATP synthase, we demonstrated that, contrary to\r\npublished data, yaku´amide inhibits both ATP hydrolysis and ATP synthesis reactions.\r\nMoreover, we found that yaku´amide inhibitory activity is proton motive force (pmf)\r\ndependent, with lower inhibition in a more coupled system. Utilising cryo-EM, we obtained\r\nmaps and models for the three main rotational states of murine ATP synthase (State 1 at 3.0 Å,\r\n8\r\nState 2 at 3.1 Å, and State 3 at 3.2 Å, overall). We observed several new features in our maps;\r\nhowever, we cannot definitively determine the exact mechanism of yaku amide’s inhibition on\r\nthe protein due to either resolution limits or suboptimal binding of the inhibitor." acknowledged_ssus: - _id: EM-Fac - _id: LifeSc alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Kristina full_name: Lukic, Kristina id: 2B04DB84-F248-11E8-B48F-1D18A9856A87 last_name: Lukic orcid: 0000-0003-1581-881X citation: ama: 'Lukic K. Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. 2024. doi:10.15479/at:ista:17319' apa: 'Lukic, K. (2024). Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:17319' chicago: 'Lukic, Kristina. “Membrane Proteins in Plant Physiology and Bioenergetics : Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the Novel Inhibitor Yaku’amide B.” Institute of Science and Technology Austria, 2024. https://doi.org/10.15479/at:ista:17319.' ieee: 'K. Lukic, “Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B,” Institute of Science and Technology Austria, 2024.' ista: 'Lukic K. 2024. Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. Institute of Science and Technology Austria.' mla: 'Lukic, Kristina. Membrane Proteins in Plant Physiology and Bioenergetics : Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the Novel Inhibitor Yaku’amide B. Institute of Science and Technology Austria, 2024, doi:10.15479/at:ista:17319.' short: 'K. Lukic, Membrane Proteins in Plant Physiology and Bioenergetics : Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the Novel Inhibitor Yaku’amide B, Institute of Science and Technology Austria, 2024.' corr_author: '1' date_created: 2024-07-26T09:05:55Z date_published: 2024-07-26T00:00:00Z date_updated: 2026-04-07T13:20:44Z day: '26' ddc: - '580' degree_awarded: PhD department: - _id: LeSa - _id: GradSch doi: 10.15479/at:ista:17319 file: - access_level: open_access checksum: 95517e697ea6a87e267e649cad560989 content_type: application/pdf creator: cchlebak date_created: 2024-07-26T13:14:24Z date_updated: 2025-01-26T23:30:04Z embargo: 2025-01-26 file_id: '17320' file_name: Thesis_Kristina_Lukic.pdf file_size: 24639084 relation: main_file - access_level: closed checksum: 74325746a9a05078fb9935dbf2aef752 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: cchlebak date_created: 2024-07-26T13:14:50Z date_updated: 2025-01-26T23:30:04Z embargo_to: open_access file_id: '17321' file_name: Thesis_Kristina_Lukic.docx file_size: 96334272 relation: source_file file_date_updated: 2025-01-26T23:30:04Z has_accepted_license: '1' language: - iso: eng month: '07' oa: 1 oa_version: Published Version page: '224' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria status: public supervisor: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 title: 'Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku''amide B' type: dissertation user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2024' ... --- _id: '12757' abstract: - lang: eng text: My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant ‘black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm (‘domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues. article_processing_charge: No article_type: review author: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: 'Sazanov LA. From the “black box” to “domino effect” mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. 2023;480(5):319-333. doi:10.1042/BCJ20210285' apa: 'Sazanov, L. A. (2023). From the “black box” to “domino effect” mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. Portland Press. https://doi.org/10.1042/BCJ20210285' chicago: 'Sazanov, Leonid A. “From the ‘black Box’ to ‘Domino Effect’ Mechanism: What Have We Learned from the Structures of Respiratory Complex I.” The Biochemical Journal. Portland Press, 2023. https://doi.org/10.1042/BCJ20210285.' ieee: 'L. A. Sazanov, “From the ‘black box’ to ‘domino effect’ mechanism: What have we learned from the structures of respiratory complex I,” The Biochemical Journal, vol. 480, no. 5. Portland Press, pp. 319–333, 2023.' ista: 'Sazanov LA. 2023. From the ‘black box’ to ‘domino effect’ mechanism: What have we learned from the structures of respiratory complex I. The Biochemical Journal. 480(5), 319–333.' mla: 'Sazanov, Leonid A. “From the ‘black Box’ to ‘Domino Effect’ Mechanism: What Have We Learned from the Structures of Respiratory Complex I.” The Biochemical Journal, vol. 480, no. 5, Portland Press, 2023, pp. 319–33, doi:10.1042/BCJ20210285.' short: L.A. Sazanov, The Biochemical Journal 480 (2023) 319–333. corr_author: '1' date_created: 2023-03-26T22:01:06Z date_published: 2023-03-15T00:00:00Z date_updated: 2024-10-09T21:04:50Z day: '15' ddc: - '570' department: - _id: LeSa doi: 10.1042/BCJ20210285 external_id: isi: - '000957065700001' pmid: - '36920092' has_accepted_license: '1' intvolume: ' 480' isi: 1 issue: '5' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1042/BCJ20210285 month: '03' oa: 1 oa_version: Published Version page: 319-333 pmid: 1 publication: The Biochemical Journal publication_identifier: eissn: - 1470-8728 issn: - 0264-6021 publication_status: published publisher: Portland Press quality_controlled: '1' scopus_import: '1' status: public title: 'From the ''black box'' to ''domino effect'' mechanism: What have we learned from the structures of respiratory complex I' tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 480 year: '2023' ... --- _id: '13232' abstract: - lang: eng text: The potential of immune-evasive mutation accumulation in the SARS-CoV-2 virus has led to its rapid spread, causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. The huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants has renewed interest in DNA vaccine technology. Here, we report the rapid generation and immunological evaluation of novel DNA vaccine candidates against the Wuhan-Hu-1 and Omicron variants based on the RBD protein fused with the Potato virus X coat protein (PVXCP). The delivery of DNA vaccines using electroporation in a two-dose regimen induced high-antibody titers and profound cellular responses in mice. The antibody titers induced against the Omicron variant of the vaccine were sufficient for effective protection against both Omicron and Wuhan-Hu-1 virus infections. The PVXCP protein in the vaccine construct shifted the immune response to the favorable Th1-like type and provided the oligomerization of RBD-PVXCP protein. Naked DNA delivery by needle-free injection allowed us to achieve antibody titers comparable with mRNA-LNP delivery in rabbits. These data identify the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study. acknowledgement: The authors declare that this study received funding from Immunofusion. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The authors express their gratitude to the Institute of Physiology of the National Academy of Sciences of Belarus for providing assistance in keeping laboratory animals. article_number: '1014' article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Mikalai full_name: Katsin, Mikalai last_name: Katsin - first_name: Maria full_name: Stegantseva, Maria last_name: Stegantseva - first_name: Sergey full_name: Golenchenko, Sergey last_name: Golenchenko - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Simon full_name: Dubovik, Simon last_name: Dubovik - first_name: Dzmitry full_name: Lutskovich, Dzmitry last_name: Lutskovich - first_name: Anton full_name: Kavaleuski, Anton id: 62304f89-eb97-11eb-a6c2-8903dd183976 last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Alexander full_name: Meleshko, Alexander last_name: Meleshko citation: ama: Dormeshkin D, Katsin M, Stegantseva M, et al. Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. 2023;11(6). doi:10.3390/vaccines11061014 apa: Dormeshkin, D., Katsin, M., Stegantseva, M., Golenchenko, S., Shapira, M., Dubovik, S., … Meleshko, A. (2023). Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. MDPI. https://doi.org/10.3390/vaccines11061014 chicago: Dormeshkin, Dmitri, Mikalai Katsin, Maria Stegantseva, Sergey Golenchenko, Michail Shapira, Simon Dubovik, Dzmitry Lutskovich, Anton Kavaleuski, and Alexander Meleshko. “Design and Immunogenicity of SARS-CoV-2 DNA Vaccine Encoding RBD-PVXCP Fusion Protein.” Vaccines. MDPI, 2023. https://doi.org/10.3390/vaccines11061014. ieee: D. Dormeshkin et al., “Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein,” Vaccines, vol. 11, no. 6. MDPI, 2023. ista: Dormeshkin D, Katsin M, Stegantseva M, Golenchenko S, Shapira M, Dubovik S, Lutskovich D, Kavaleuski A, Meleshko A. 2023. Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein. Vaccines. 11(6), 1014. mla: Dormeshkin, Dmitri, et al. “Design and Immunogenicity of SARS-CoV-2 DNA Vaccine Encoding RBD-PVXCP Fusion Protein.” Vaccines, vol. 11, no. 6, 1014, MDPI, 2023, doi:10.3390/vaccines11061014. short: D. Dormeshkin, M. Katsin, M. Stegantseva, S. Golenchenko, M. Shapira, S. Dubovik, D. Lutskovich, A. Kavaleuski, A. Meleshko, Vaccines 11 (2023). date_created: 2023-07-16T22:01:10Z date_published: 2023-06-01T00:00:00Z date_updated: 2025-04-23T13:01:23Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.3390/vaccines11061014 external_id: isi: - '001017740000001' pmid: - '37376403' file: - access_level: open_access checksum: 8f484c0f30f8699c589b1c29a0fd7d7f content_type: application/pdf creator: dernst date_created: 2023-07-18T07:25:43Z date_updated: 2023-07-18T07:25:43Z file_id: '13244' file_name: 2023_Vaccines_Dormeshkin.pdf file_size: 2339746 relation: main_file success: 1 file_date_updated: 2023-07-18T07:25:43Z has_accepted_license: '1' intvolume: ' 11' isi: 1 issue: '6' language: - iso: eng month: '06' oa: 1 oa_version: Published Version pmid: 1 publication: Vaccines publication_identifier: eissn: - 2076-393X publication_status: published publisher: MDPI quality_controlled: '1' scopus_import: '1' status: public title: Design and immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 11 year: '2023' ... --- _id: '14040' abstract: - lang: eng text: Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster. acknowledged_ssus: - _id: EM-Fac - _id: LifeSc - _id: ScienComp acknowledgement: P.J.N. and J.W.M. are grateful for the support of the Biotechnology & Biological Sciences Research Council (awards BB/L003260/1 and BB/P00931X/1). J. Knoppová, R.S. and J. Komenda were supported by the Czech Science Foundation (project 19-29225X) and by ERC project Photoredesign (no. 854126) and L.A.S. was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Electron Microscopy Facility (EMF), the Life Science Facility (LSF) and the IST high-performance computing cluster. article_number: '4681' article_processing_charge: Yes article_type: original author: - first_name: Ziyu full_name: Zhao, Ziyu last_name: Zhao - first_name: Irene full_name: Vercellino, Irene id: 3ED6AF16-F248-11E8-B48F-1D18A9856A87 last_name: Vercellino orcid: 0000-0001-5618-3449 - first_name: Jana full_name: Knoppová, Jana last_name: Knoppová - first_name: Roman full_name: Sobotka, Roman last_name: Sobotka - first_name: James W. full_name: Murray, James W. last_name: Murray - first_name: Peter J. full_name: Nixon, Peter J. last_name: Nixon - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 - first_name: Josef full_name: Komenda, Josef last_name: Komenda citation: ama: Zhao Z, Vercellino I, Knoppová J, et al. The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. 2023;14. doi:10.1038/s41467-023-40388-6 apa: Zhao, Z., Vercellino, I., Knoppová, J., Sobotka, R., Murray, J. W., Nixon, P. J., … Komenda, J. (2023). The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-023-40388-6 chicago: Zhao, Ziyu, Irene Vercellino, Jana Knoppová, Roman Sobotka, James W. Murray, Peter J. Nixon, Leonid A Sazanov, and Josef Komenda. “The Ycf48 Accessory Factor Occupies the Site of the Oxygen-Evolving Manganese Cluster during Photosystem II Biogenesis.” Nature Communications. Springer Nature, 2023. https://doi.org/10.1038/s41467-023-40388-6. ieee: Z. Zhao et al., “The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis,” Nature Communications, vol. 14. Springer Nature, 2023. ista: Zhao Z, Vercellino I, Knoppová J, Sobotka R, Murray JW, Nixon PJ, Sazanov LA, Komenda J. 2023. The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis. Nature Communications. 14, 4681. mla: Zhao, Ziyu, et al. “The Ycf48 Accessory Factor Occupies the Site of the Oxygen-Evolving Manganese Cluster during Photosystem II Biogenesis.” Nature Communications, vol. 14, 4681, Springer Nature, 2023, doi:10.1038/s41467-023-40388-6. short: Z. Zhao, I. Vercellino, J. Knoppová, R. Sobotka, J.W. Murray, P.J. Nixon, L.A. Sazanov, J. Komenda, Nature Communications 14 (2023). corr_author: '1' date_created: 2023-08-13T22:01:13Z date_published: 2023-08-04T00:00:00Z date_updated: 2025-04-23T13:05:33Z day: '04' ddc: - '570' department: - _id: LeSa doi: 10.1038/s41467-023-40388-6 external_id: isi: - '001042606700004' pmid: - '37542031' file: - access_level: open_access checksum: 3b9043df3d51c300f9be95eac3ff9d0b content_type: application/pdf creator: dernst date_created: 2023-08-14T07:01:12Z date_updated: 2023-08-14T07:01:12Z file_id: '14044' file_name: 2023_NatureComm_Zhao.pdf file_size: 2315325 relation: main_file success: 1 file_date_updated: 2023-08-14T07:01:12Z has_accepted_license: '1' intvolume: ' 14' isi: 1 language: - iso: eng month: '08' oa: 1 oa_version: Published Version pmid: 1 publication: Nature Communications publication_identifier: eissn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 14 year: '2023' ... --- OA_place: publisher _id: '12781' abstract: - lang: eng text: "Most energy in humans is produced in form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). Complex I is the first and the largest enzyme of the respiratory chain which is essential for energy production. It couples the transfer of two electrons from NADH to ubiquinone with proton translocation across bacterial or inner mitochondrial membrane. The coupling mechanism between electron transfer and proton translocation is one of the biggest enigma in bioenergetics and structural biology. Even though the enzyme has been studied for decades, only recent technological advances in cryo-EM allowed its extensive structural investigation. \r\n\r\nComplex I from E.coli appears to be of special importance because it is a perfect model system with a rich mutant library, however the structure of the entire complex was unknown. In this thesis I have resolved structures of the minimal complex I version from E. coli in different states including reduced, inhibited, under reaction turnover and several others. Extensive structural analyses of these structures and comparison to structures from other species allowed to derive general features of conformational dynamics and propose a universal coupling mechanism. The mechanism is straightforward, robust and consistent with decades of experimental data available for complex I from different species. \r\n\r\nCyanobacterial NDH (cyanobacterial complex I) is a part of broad complex I superfamily and was studied as well in this thesis. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Here, I solved structure of NDH and revealed additional state, which was not observed before. The novel “resting” state allowed to propose the mechanism of CET regulation. Moreover, conformational dynamics of NDH resembles one in complex I which suggest more broad universality of the proposed coupling mechanism.\r\n\r\nIn summary, results presented here helped to interpret decades of experimental data for complex I and contributed to fundamental mechanistic understanding of protein function.\r\n" acknowledged_ssus: - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Vladyslav full_name: Kravchuk, Vladyslav id: 4D62F2A6-F248-11E8-B48F-1D18A9856A87 last_name: Kravchuk orcid: 0000-0001-9523-9089 citation: ama: Kravchuk V. Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. 2023. doi:10.15479/at:ista:12781 apa: Kravchuk, V. (2023). Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:12781 chicago: Kravchuk, Vladyslav. “Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog.” Institute of Science and Technology Austria, 2023. https://doi.org/10.15479/at:ista:12781. ieee: V. Kravchuk, “Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog,” Institute of Science and Technology Austria, 2023. ista: Kravchuk V. 2023. Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog. Institute of Science and Technology Austria. mla: Kravchuk, Vladyslav. Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog. Institute of Science and Technology Austria, 2023, doi:10.15479/at:ista:12781. short: V. Kravchuk, Structural and Mechanistic Study of Bacterial Complex I and Its Cyanobacterial Ortholog, Institute of Science and Technology Austria, 2023. corr_author: '1' date_created: 2023-03-31T12:24:42Z date_published: 2023-03-23T00:00:00Z date_updated: 2026-04-07T14:10:40Z day: '23' ddc: - '570' - '572' degree_awarded: PhD department: - _id: GradSch - _id: LeSa doi: 10.15479/at:ista:12781 ec_funded: 1 file: - access_level: open_access checksum: 5ebb6345cb4119f93460c81310265a6d content_type: application/pdf creator: vkravchu date_created: 2023-04-19T14:33:41Z date_updated: 2024-04-22T22:30:06Z embargo: 2024-04-20 file_id: '12852' file_name: VladyslavKravchuk_PhD_Thesis_PostSub_Final_1.pdf file_size: 6071553 relation: main_file - access_level: open_access checksum: c12055c48411d030d2afa51de2166221 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: vkravchu date_created: 2023-04-19T14:33:52Z date_updated: 2024-04-22T22:30:06Z embargo: 2024-04-20 file_id: '12853' file_name: VladyslavKravchuk_PhD_Thesis_PostSub_Final.docx file_size: 19468766 relation: source_file file_date_updated: 2024-04-22T22:30:06Z has_accepted_license: '1' language: - iso: eng month: '03' oa: 1 oa_version: Published Version page: '127' project: - _id: 238A0A5A-32DE-11EA-91FC-C7463DDC885E grant_number: '25541' name: 'Structural characterization of E. coli complex I: an important mechanistic model' - _id: 627abdeb-2b32-11ec-9570-ec31a97243d3 call_identifier: H2020 grant_number: '101020697' name: Structure and mechanism of respiratory chain molecular machines publication_identifier: isbn: - 978-3-99078-029-9 issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '12138' relation: part_of_dissertation status: public status: public supervisor: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 title: Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog type: dissertation user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2023' ... --- DOAJ_listed: '1' OA_place: publisher OA_type: gold _id: '10945' abstract: - lang: eng text: Mica-titania pearlescent pigments (MTs) were previously coated with organic molecules to obtain combination pigments (CPs) for achieving certain improvements or functionalities. Anthocyanins (ACNs) are molecules that can be extracted from natural resources and exhibit color changes via pH modifications of the enclosing medium. The purpose of the study was to produce a new series of CPs by depositing ACNs on MTs at different pH values, to observe the changes in color, and to associate these changes to thermogravimetrically determined deposition efficiencies in light of spectral differences. The extraction and deposition methods were based on aqueous chemistry and were straightforward. The ACN deposition generally increased with increasing pH and correlated with the consistency between the charges of the MT surfaces and the dominant ACN species at a specific pH value. The fluorescence of the CPs was inversely correlated with the deposition quantities invoking the possibility of a quenching effect. acknowledgement: "This research was partly funded by Hacettepe University (Bilimsel Ara¸stırma Projeleri\r\nKoordinasyon Birimi), grant number FHD-2015-8094.The authors are indebted to Ahmet Önal for his supports in acquiring the fluorescence spectra and the decision of excitation wavelengths. The authors also acknowledge use of the services and facilities of UNAM-National Nanotechnology Research Center at Bilkent University and mica donation from Sabuncular Mining Co." article_processing_charge: Yes article_type: original author: - first_name: Mehmet Orkun full_name: Çoruh, Mehmet Orkun id: d25163e5-8d53-11eb-a251-e6dd8ea1b8ef last_name: Çoruh orcid: 0000-0002-3219-2022 - first_name: Güngör full_name: Gündüz, Güngör last_name: Gündüz - first_name: Üner full_name: Çolak, Üner last_name: Çolak - first_name: Bora full_name: Maviş, Bora last_name: Maviş citation: ama: Çoruh MO, Gündüz G, Çolak Ü, Maviş B. pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra. Colorants. 2022;1(2):149-164. doi:10.3390/colorants1020010 apa: Çoruh, M. O., Gündüz, G., Çolak, Ü., & Maviş, B. (2022). pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra. Colorants. MDPI. https://doi.org/10.3390/colorants1020010 chicago: Çoruh, Mehmet Orkun, Güngör Gündüz, Üner Çolak, and Bora Maviş. “PH-Dependent Coloring of Combination Effect Pigments with Anthocyanins from Brassica Oleracea Var. Capitata F. Rubra.” Colorants. MDPI, 2022. https://doi.org/10.3390/colorants1020010. ieee: M. O. Çoruh, G. Gündüz, Ü. Çolak, and B. Maviş, “pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra,” Colorants, vol. 1, no. 2. MDPI, pp. 149–164, 2022. ista: Çoruh MO, Gündüz G, Çolak Ü, Maviş B. 2022. pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra. Colorants. 1(2), 149–164. mla: Çoruh, Mehmet Orkun, et al. “PH-Dependent Coloring of Combination Effect Pigments with Anthocyanins from Brassica Oleracea Var. Capitata F. Rubra.” Colorants, vol. 1, no. 2, MDPI, 2022, pp. 149–64, doi:10.3390/colorants1020010. short: M.O. Çoruh, G. Gündüz, Ü. Çolak, B. Maviş, Colorants 1 (2022) 149–164. date_created: 2022-04-04T09:03:54Z date_published: 2022-04-01T00:00:00Z date_updated: 2024-10-14T13:52:09Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.3390/colorants1020010 file: - access_level: open_access checksum: 2c15c8d3041ebc36bc64870247081758 content_type: application/pdf creator: dernst date_created: 2022-04-04T10:39:24Z date_updated: 2022-04-04T10:39:24Z file_id: '10949' file_name: 2022_Colorants_Coruh.pdf file_size: 2437988 relation: main_file success: 1 file_date_updated: 2022-04-04T10:39:24Z has_accepted_license: '1' intvolume: ' 1' issue: '2' language: - iso: eng month: '04' oa: 1 oa_version: Published Version page: 149-164 publication: Colorants publication_identifier: issn: - 2079-6447 publication_status: published publisher: MDPI quality_controlled: '1' status: public title: pH-dependent coloring of combination effect pigments with anthocyanins from Brassica oleracea var. capitata F. rubra tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 0043cee0-e5fc-11ee-9736-f83bc23afbf0 volume: 1 year: '2022' ... --- _id: '11167' abstract: - lang: eng text: Complex I is one of the major respiratory complexes, conserved from bacteria to mammals. It oxidises NADH, reduces quinone and pumps protons across the membrane, thus playing a central role in the oxidative energy metabolism. In this review we discuss our current state of understanding the structure of complex I from various species of mammals, plants, fungi, and bacteria, as well as of several complex I-related proteins. By comparing the structural evidence from these systems in different redox states and data from mutagenesis and molecular simulations, we formulate the mechanisms of electron transfer and proton pumping and explain how they are conformationally and electrostatically coupled. Finally, we discuss the structural basis of the deactivation phenomenon in mammalian complex I. article_number: '102350' article_processing_charge: Yes (via OA deal) article_type: original author: - first_name: Domen full_name: Kampjut, Domen id: 37233050-F248-11E8-B48F-1D18A9856A87 last_name: Kampjut - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Kampjut D, Sazanov LA. Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. 2022;74. doi:10.1016/j.sbi.2022.102350 apa: Kampjut, D., & Sazanov, L. A. (2022). Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. Elsevier. https://doi.org/10.1016/j.sbi.2022.102350 chicago: Kampjut, Domen, and Leonid A Sazanov. “Structure of Respiratory Complex I – An Emerging Blueprint for the Mechanism.” Current Opinion in Structural Biology. Elsevier, 2022. https://doi.org/10.1016/j.sbi.2022.102350. ieee: D. Kampjut and L. A. Sazanov, “Structure of respiratory complex I – An emerging blueprint for the mechanism,” Current Opinion in Structural Biology, vol. 74. Elsevier, 2022. ista: Kampjut D, Sazanov LA. 2022. Structure of respiratory complex I – An emerging blueprint for the mechanism. Current Opinion in Structural Biology. 74, 102350. mla: Kampjut, Domen, and Leonid A. Sazanov. “Structure of Respiratory Complex I – An Emerging Blueprint for the Mechanism.” Current Opinion in Structural Biology, vol. 74, 102350, Elsevier, 2022, doi:10.1016/j.sbi.2022.102350. short: D. Kampjut, L.A. Sazanov, Current Opinion in Structural Biology 74 (2022). corr_author: '1' date_created: 2022-04-15T09:32:35Z date_published: 2022-06-01T00:00:00Z date_updated: 2024-10-09T21:02:00Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1016/j.sbi.2022.102350 external_id: isi: - '000829029500020' pmid: - '35316665' file: - access_level: open_access checksum: 72bdde48853643a32d42b75f54965c44 content_type: application/pdf creator: dernst date_created: 2022-08-05T05:56:03Z date_updated: 2022-08-05T05:56:03Z file_id: '11725' file_name: 2022_CurrentOpStructBiology_Kampjut.pdf file_size: 815607 relation: main_file success: 1 file_date_updated: 2022-08-05T05:56:03Z has_accepted_license: '1' intvolume: ' 74' isi: 1 keyword: - Molecular Biology - Structural Biology language: - iso: eng month: '06' oa: 1 oa_version: Published Version pmid: 1 publication: Current Opinion in Structural Biology publication_identifier: issn: - 0959-440X publication_status: published publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: Structure of respiratory complex I – An emerging blueprint for the mechanism tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 74 year: '2022' ... --- _id: '11462' abstract: - lang: eng text: Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. acknowledgement: This study was financially supported by the State Committee on Science and Technology. We would like to thank Elena Tumar and Elena Kisileva at the Institute of Bioorganic Chemistry of NASB for their kind assistance with mouse immunizations. article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Alena full_name: Karputs, Alena last_name: Karputs - first_name: Anton full_name: Kavaleuski, Anton id: 62304f89-eb97-11eb-a6c2-8903dd183976 last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Ivan full_name: Kuzminski, Ivan last_name: Kuzminski - first_name: Elena full_name: Stepanova, Elena last_name: Stepanova - first_name: Andrei full_name: Gilep, Andrei last_name: Gilep citation: ama: Dormeshkin D, Shapira M, Karputs A, et al. Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. 2022;106:5093-5103. doi:10.1007/s00253-022-12022-w apa: Dormeshkin, D., Shapira, M., Karputs, A., Kavaleuski, A., Kuzminski, I., Stepanova, E., & Gilep, A. (2022). Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. Springer Nature. https://doi.org/10.1007/s00253-022-12022-w chicago: Dormeshkin, Dmitri, Michail Shapira, Alena Karputs, Anton Kavaleuski, Ivan Kuzminski, Elena Stepanova, and Andrei Gilep. “Combining of Synthetic VHH and Immune ScFv Libraries for Pregnancy-Associated Glycoproteins ELISA Development.” Applied Microbiology and Biotechnology. Springer Nature, 2022. https://doi.org/10.1007/s00253-022-12022-w. ieee: D. Dormeshkin et al., “Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development,” Applied Microbiology and Biotechnology, vol. 106. Springer Nature, pp. 5093–5103, 2022. ista: Dormeshkin D, Shapira M, Karputs A, Kavaleuski A, Kuzminski I, Stepanova E, Gilep A. 2022. Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. 106, 5093–5103. mla: Dormeshkin, Dmitri, et al. “Combining of Synthetic VHH and Immune ScFv Libraries for Pregnancy-Associated Glycoproteins ELISA Development.” Applied Microbiology and Biotechnology, vol. 106, Springer Nature, 2022, pp. 5093–103, doi:10.1007/s00253-022-12022-w. short: D. Dormeshkin, M. Shapira, A. Karputs, A. Kavaleuski, I. Kuzminski, E. Stepanova, A. Gilep, Applied Microbiology and Biotechnology 106 (2022) 5093–5103. date_created: 2022-06-26T22:01:34Z date_published: 2022-08-01T00:00:00Z date_updated: 2023-10-10T07:15:02Z day: '01' department: - _id: GradSch - _id: LeSa doi: 10.1007/s00253-022-12022-w external_id: isi: - '000813677500001' pmid: - '35723693' intvolume: ' 106' isi: 1 language: - iso: eng month: '08' oa_version: None page: 5093-5103 pmid: 1 publication: Applied Microbiology and Biotechnology publication_identifier: eissn: - 1432-0614 issn: - 0175-7598 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 106 year: '2022' ... --- _id: '11551' abstract: - lang: eng text: Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance. acknowledgement: "We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459).\r\n" article_number: '620' article_processing_charge: No author: - first_name: David full_name: Molina-Granada, David last_name: Molina-Granada - first_name: Emiliano full_name: González-Vioque, Emiliano last_name: González-Vioque - first_name: Marris G. full_name: Dibley, Marris G. last_name: Dibley - first_name: Raquel full_name: Cabrera-Pérez, Raquel last_name: Cabrera-Pérez - first_name: Antoni full_name: Vallbona-Garcia, Antoni last_name: Vallbona-Garcia - first_name: Javier full_name: Torres-Torronteras, Javier last_name: Torres-Torronteras - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 - first_name: Michael T. full_name: Ryan, Michael T. last_name: Ryan - first_name: Yolanda full_name: Cámara, Yolanda last_name: Cámara - first_name: Ramon full_name: Martí, Ramon last_name: Martí citation: ama: Molina-Granada D, González-Vioque E, Dibley MG, et al. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. 2022;5(1). doi:10.1038/s42003-022-03568-6 apa: Molina-Granada, D., González-Vioque, E., Dibley, M. G., Cabrera-Pérez, R., Vallbona-Garcia, A., Torres-Torronteras, J., … Martí, R. (2022). Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. Springer Nature. https://doi.org/10.1038/s42003-022-03568-6 chicago: Molina-Granada, David, Emiliano González-Vioque, Marris G. Dibley, Raquel Cabrera-Pérez, Antoni Vallbona-Garcia, Javier Torres-Torronteras, Leonid A Sazanov, Michael T. Ryan, Yolanda Cámara, and Ramon Martí. “Most Mitochondrial DGTP Is Tightly Bound to Respiratory Complex I through the NDUFA10 Subunit.” Communications Biology. Springer Nature, 2022. https://doi.org/10.1038/s42003-022-03568-6. ieee: D. Molina-Granada et al., “Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit,” Communications Biology, vol. 5, no. 1. Springer Nature, 2022. ista: Molina-Granada D, González-Vioque E, Dibley MG, Cabrera-Pérez R, Vallbona-Garcia A, Torres-Torronteras J, Sazanov LA, Ryan MT, Cámara Y, Martí R. 2022. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit. Communications Biology. 5(1), 620. mla: Molina-Granada, David, et al. “Most Mitochondrial DGTP Is Tightly Bound to Respiratory Complex I through the NDUFA10 Subunit.” Communications Biology, vol. 5, no. 1, 620, Springer Nature, 2022, doi:10.1038/s42003-022-03568-6. short: D. Molina-Granada, E. González-Vioque, M.G. Dibley, R. Cabrera-Pérez, A. Vallbona-Garcia, J. Torres-Torronteras, L.A. Sazanov, M.T. Ryan, Y. Cámara, R. Martí, Communications Biology 5 (2022). date_created: 2022-07-10T22:01:52Z date_published: 2022-06-23T00:00:00Z date_updated: 2026-04-02T13:22:53Z day: '23' ddc: - '570' department: - _id: LeSa doi: 10.1038/s42003-022-03568-6 external_id: isi: - '000815098500002' pmid: - ' 35739187' file: - access_level: open_access checksum: 965f88bbcef3fd0c3e121340555c4467 content_type: application/pdf creator: kschuh date_created: 2022-07-13T07:44:58Z date_updated: 2022-07-13T07:44:58Z file_id: '11571' file_name: 2022_communicationsbiology_Molina-Granada.pdf file_size: 2335369 relation: main_file success: 1 file_date_updated: 2022-07-13T07:44:58Z has_accepted_license: '1' intvolume: ' 5' isi: 1 issue: '1' language: - iso: eng month: '06' oa: 1 oa_version: Published Version pmid: 1 publication: Communications Biology publication_identifier: eissn: - 2399-3642 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd volume: 5 year: '2022' ... --- _id: '11648' abstract: - lang: eng text: 'Progress in structural membrane biology has been significantly accelerated by the ongoing ''Resolution Revolution'' in cryo electron microscopy (cryo-EM). In particular, structure determination by single particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor LP-ring from Salmonella enterica.' acknowledgement: "Cyclic Innovation for Clinical Empowerment (JP17pc0101020 from Japan Agency for Medical Research and Development (AMED) to K.N. and G.K.); Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED (JP20am0101117 to K.N., JP16K07266 to Atsunori Oshima and C.G., JP22ama121001j0001 to Masaki Yamamoto, G.K., T.K. and C.G.); a JSPS KAHKENHI\r\ngrant (20K06514 to J.K.) and a Grant-in-aid for JSPS fellows (20J00162 to A.N.).\r\nWe are grateful for initiation and scientific support from Matthias Rogner, Marc M. Nowaczyk, Anna Frank and ̈Yuko Misumi for the PSI monomer project and also would like to thank Hideki Shigematsu for critical reading of the manuscript. And we are indebted to the two anonymous reviewers who helped us to improve our manuscript." article_processing_charge: No article_type: original author: - first_name: Christoph full_name: Gerle, Christoph last_name: Gerle - first_name: Jun-ichi full_name: Kishikawa, Jun-ichi last_name: Kishikawa - first_name: Tomoko full_name: Yamaguchi, Tomoko last_name: Yamaguchi - first_name: Atsuko full_name: Nakanishi, Atsuko last_name: Nakanishi - first_name: Mehmet Orkun full_name: Çoruh, Mehmet Orkun id: d25163e5-8d53-11eb-a251-e6dd8ea1b8ef last_name: Çoruh orcid: 0000-0002-3219-2022 - first_name: Fumiaki full_name: Makino, Fumiaki last_name: Makino - first_name: Tomoko full_name: Miyata, Tomoko last_name: Miyata - first_name: Akihiro full_name: Kawamoto, Akihiro last_name: Kawamoto - first_name: Ken full_name: Yokoyama, Ken last_name: Yokoyama - first_name: Keiichi full_name: Namba, Keiichi last_name: Namba - first_name: Genji full_name: Kurisu, Genji last_name: Kurisu - first_name: Takayuki full_name: Kato, Takayuki last_name: Kato citation: ama: Gerle C, Kishikawa J, Yamaguchi T, et al. Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. 2022;71(5):249-261. doi:10.1093/jmicro/dfac037 apa: Gerle, C., Kishikawa, J., Yamaguchi, T., Nakanishi, A., Çoruh, M. O., Makino, F., … Kato, T. (2022). Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. Oxford University Press. https://doi.org/10.1093/jmicro/dfac037 chicago: Gerle, Christoph, Jun-ichi Kishikawa, Tomoko Yamaguchi, Atsuko Nakanishi, Mehmet Orkun Çoruh, Fumiaki Makino, Tomoko Miyata, et al. “Structures of Multisubunit Membrane Complexes with the CRYO ARM 200.” Microscopy. Oxford University Press, 2022. https://doi.org/10.1093/jmicro/dfac037. ieee: C. Gerle et al., “Structures of multisubunit membrane complexes with the CRYO ARM 200,” Microscopy, vol. 71, no. 5. Oxford University Press, pp. 249–261, 2022. ista: Gerle C, Kishikawa J, Yamaguchi T, Nakanishi A, Çoruh MO, Makino F, Miyata T, Kawamoto A, Yokoyama K, Namba K, Kurisu G, Kato T. 2022. Structures of multisubunit membrane complexes with the CRYO ARM 200. Microscopy. 71(5), 249–261. mla: Gerle, Christoph, et al. “Structures of Multisubunit Membrane Complexes with the CRYO ARM 200.” Microscopy, vol. 71, no. 5, Oxford University Press, 2022, pp. 249–61, doi:10.1093/jmicro/dfac037. short: C. Gerle, J. Kishikawa, T. Yamaguchi, A. Nakanishi, M.O. Çoruh, F. Makino, T. Miyata, A. Kawamoto, K. Yokoyama, K. Namba, G. Kurisu, T. Kato, Microscopy 71 (2022) 249–261. date_created: 2022-07-25T10:04:58Z date_published: 2022-10-01T00:00:00Z date_updated: 2023-08-03T12:13:37Z day: '01' ddc: - '570' department: - _id: LeSa doi: 10.1093/jmicro/dfac037 external_id: isi: - '000837950900001' pmid: - '35861182' file: - access_level: open_access checksum: 23b51c163636bf9313f7f0818312e67e content_type: application/pdf creator: dernst date_created: 2023-02-03T08:34:48Z date_updated: 2023-02-03T08:34:48Z file_id: '12498' file_name: 2022_Microscopy_Gerle.pdf file_size: 7812696 relation: main_file success: 1 file_date_updated: 2023-02-03T08:34:48Z has_accepted_license: '1' intvolume: ' 71' isi: 1 issue: '5' keyword: - Radiology - Nuclear Medicine and imaging - Instrumentation - Structural Biology language: - iso: eng month: '10' oa: 1 oa_version: Published Version page: 249-261 pmid: 1 publication: Microscopy publication_identifier: eissn: - 2050-5701 issn: - 2050-5698 publication_status: published publisher: Oxford University Press quality_controlled: '1' scopus_import: '1' status: public title: Structures of multisubunit membrane complexes with the CRYO ARM 200 tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 71 year: '2022' ... --- _id: '12252' abstract: - lang: eng text: The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants. acknowledgement: The authors declare that this study received funding from Immunofusion. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. article_number: '965446' article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Simon full_name: Dubovik, Simon last_name: Dubovik - first_name: Anton full_name: Kavaleuski, Anton id: 4968f7ad-eb97-11eb-a6c2-8ed382e8912c last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Mikalai full_name: Katsin, Mikalai last_name: Katsin - first_name: Alexandr full_name: Migas, Alexandr last_name: Migas - first_name: Alexander full_name: Meleshko, Alexander last_name: Meleshko - first_name: Sergei full_name: Semyonov, Sergei last_name: Semyonov citation: ama: Dormeshkin D, Shapira M, Dubovik S, et al. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 2022;13. doi:10.3389/fimmu.2022.965446 apa: Dormeshkin, D., Shapira, M., Dubovik, S., Kavaleuski, A., Katsin, M., Migas, A., … Semyonov, S. (2022). Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. Frontiers Media. https://doi.org/10.3389/fimmu.2022.965446 chicago: Dormeshkin, Dmitri, Michail Shapira, Simon Dubovik, Anton Kavaleuski, Mikalai Katsin, Alexandr Migas, Alexander Meleshko, and Sergei Semyonov. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology. Frontiers Media, 2022. https://doi.org/10.3389/fimmu.2022.965446. ieee: D. Dormeshkin et al., “Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library,” Frontiers in Immunology, vol. 13. Frontiers Media, 2022. ista: Dormeshkin D, Shapira M, Dubovik S, Kavaleuski A, Katsin M, Migas A, Meleshko A, Semyonov S. 2022. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 13, 965446. mla: Dormeshkin, Dmitri, et al. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology, vol. 13, 965446, Frontiers Media, 2022, doi:10.3389/fimmu.2022.965446. short: D. Dormeshkin, M. Shapira, S. Dubovik, A. Kavaleuski, M. Katsin, A. Migas, A. Meleshko, S. Semyonov, Frontiers in Immunology 13 (2022). date_created: 2023-01-16T09:56:57Z date_published: 2022-09-16T00:00:00Z date_updated: 2025-06-11T13:42:26Z day: '16' ddc: - '570' department: - _id: LeSa doi: 10.3389/fimmu.2022.965446 external_id: isi: - '000862479100001' pmid: - '36189235' file: - access_level: open_access checksum: f8f5d8110710033d0532e7e08bf9dad4 content_type: application/pdf creator: dernst date_created: 2023-01-30T09:22:26Z date_updated: 2023-01-30T09:22:26Z file_id: '12443' file_name: 2022_FrontiersImmunology_Dormeshkin.pdf file_size: 5695892 relation: main_file success: 1 file_date_updated: 2023-01-30T09:22:26Z has_accepted_license: '1' intvolume: ' 13' isi: 1 keyword: - Immunology - Immunology and Allergy - COVID-19 - SARS-CoV-2 - synthetic library - RBD - neutralization nanobody - VHH language: - iso: eng month: '09' oa: 1 oa_version: Published Version pmid: 1 publication: Frontiers in Immunology publication_identifier: issn: - 1664-3224 publication_status: published publisher: Frontiers Media quality_controlled: '1' scopus_import: '1' status: public title: Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 13 year: '2022' ... --- _id: '12282' abstract: - lang: eng text: From a simple thought to a multicellular movement acknowledgement: The authors want to thank Professors Carrie Bernecky, Tom Henzinger, Martin Loose and Gaia Novarino for accepting to be interviewed, thus giving significant contribution to the discussion that lead to this article. article_number: '260017' article_processing_charge: No article_type: letter_note author: - first_name: Nicole full_name: Amberg, Nicole id: 4CD6AAC6-F248-11E8-B48F-1D18A9856A87 last_name: Amberg orcid: 0000-0002-3183-8207 - first_name: Melissa A full_name: Stouffer, Melissa A id: 4C9372C4-F248-11E8-B48F-1D18A9856A87 last_name: Stouffer - first_name: Irene full_name: Vercellino, Irene id: 3ED6AF16-F248-11E8-B48F-1D18A9856A87 last_name: Vercellino orcid: 0000-0001-5618-3449 citation: ama: Amberg N, Stouffer MA, Vercellino I. Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole. Journal of Cell Science. 2022;135(8). doi:10.1242/jcs.260017 apa: Amberg, N., Stouffer, M. A., & Vercellino, I. (2022). Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole. Journal of Cell Science. The Company of Biologists. https://doi.org/10.1242/jcs.260017 chicago: Amberg, Nicole, Melissa A Stouffer, and Irene Vercellino. “Operation STEM Fatale – How an Equity, Diversity and Inclusion Initiative Has Brought Us to Reflect on the Current Challenges in Cell Biology and Science as a Whole.” Journal of Cell Science. The Company of Biologists, 2022. https://doi.org/10.1242/jcs.260017. ieee: N. Amberg, M. A. Stouffer, and I. Vercellino, “Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole,” Journal of Cell Science, vol. 135, no. 8. The Company of Biologists, 2022. ista: Amberg N, Stouffer MA, Vercellino I. 2022. Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole. Journal of Cell Science. 135(8), 260017. mla: Amberg, Nicole, et al. “Operation STEM Fatale – How an Equity, Diversity and Inclusion Initiative Has Brought Us to Reflect on the Current Challenges in Cell Biology and Science as a Whole.” Journal of Cell Science, vol. 135, no. 8, 260017, The Company of Biologists, 2022, doi:10.1242/jcs.260017. short: N. Amberg, M.A. Stouffer, I. Vercellino, Journal of Cell Science 135 (2022). corr_author: '1' date_created: 2023-01-16T10:03:14Z date_published: 2022-04-19T00:00:00Z date_updated: 2024-10-09T21:03:55Z day: '19' department: - _id: SiHi - _id: LeSa doi: 10.1242/jcs.260017 external_id: isi: - '000798123600015' pmid: - '35438168' intvolume: ' 135' isi: 1 issue: '8' language: - iso: eng month: '04' oa_version: None pmid: 1 publication: Journal of Cell Science publication_identifier: eissn: - 1477-9137 issn: - 0021-9533 publication_status: published publisher: The Company of Biologists quality_controlled: '1' scopus_import: '1' status: public title: Operation STEM fatale – how an equity, diversity and inclusion initiative has brought us to reflect on the current challenges in cell biology and science as a whole type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 135 year: '2022' ...