---
OA_place: publisher
OA_type: hybrid
PlanS_conform: '1'
_id: '20430'
abstract:
- lang: eng
  text: Protein design has focused on the design of ground states, ensuring that they
    are sufficiently low energy to be highly populated1. Designing the kinetics and
    dynamics of a system requires, in addition, the design of excited states that
    are traversed in transitions from one low-lying state to another2,3. This is a
    challenging task because such states must be sufficiently strained to be poorly
    populated, but not so strained that they are not populated at all, and because
    protein design methods have focused on generating near-ideal structures4,5,6,7.
    Here we describe a general approach for designing systems that use an induced-fit
    power stroke8 to generate a structurally frustrated9 and strained excited state,
    allosterically driving protein complex dissociation. X-ray crystallography, double
    electron–electron resonance spectroscopy and kinetic binding measurements show
    that incorporating excited states enables the design of effector-induced increases
    in dissociation rates as high as 5,700-fold. We highlight the power of this approach
    by designing rapid biosensors, kinetically controlled circuits and cytokine mimics
    that can be dissociated from their receptors within seconds, enabling dissection
    of the temporal dynamics of interleukin-2 signalling.
acknowledgement: We thank P. J. Y. Leung, K. L. Shelley, A. Pillai, C. Demakis, M.
  Exposit, K. Thompson, C. Savvides, R. J. Ragotte, G. Ahn and M. Glögl for discussions
  and technical support; K. VanWormer and L. Goldschmidt for technical support; S.
  R. Gerben and A. Murray for protein production support; and X. Li, M. Lamb, Z. Taylor
  and V. Adebomi for LC–MS support. This work was supported by the Audacious Project
  at the Institute for Protein Design (A.J.B., A.K., J.D.L.C., E.B. and A.K.B.); by
  a gift from Microsoft (A.J.B.); by the Nordstrom Barrier Institute for Protein Design
  Directors Fund (M.H.A. and F.P.); by Bill and Melinda Gates Foundation OPP1156262
  (A.K. and J.D.L.C.); by the Open Philanthropy Project Improving Protein Design Fund
  (E.B. and A.K.B.); by the National Institutes of Health (NIH) National Institute
  of Allergy and Infectious Disease grant R0AI160052 (A.K.B.); by CRI Irvington Postdoctoral
  Fellowship 315511 (Y.Z.); by National Cancer Institute K00 award 4K00CA274708 (M.O.);
  by National Science Foundation grant MCB 2119837 and NIH grant GM115805 (W.H.R.
  and D.M.Z.); by NIH grant GM151956 (S.S.); by NIH AI-51321 (K.C.G.); by the DFG
  grants PI 405/15 and SFB 1557 (C.P. and J.P.); and by the Howard Hughes Medical
  Institute (A.K.B., K.C.G. and D.B.). The EPR spectrometer used for the DEER experiments
  was in part supported by NIH grant S10OD021557. This research used resources (FMX/AMX)
  of the National Synchrotron Light Source II, a US Department of Energy (DoE) Office
  of Science User Facility operated for the DOE Office of Science by Brookhaven National
  Laboratory under contract DE-SC0012704. The Center for BioMolecular Structure (CBMS)
  is supported mainly by the NIH National Institute of General Medical Sciences (NIGMS)
  through a Center Core P30 Grant (P30GM133893), and by the DoE Office of Biological
  and Environmental Research (KP1607011). This work is based on research performed
  at the Northeastern Collaborative Access Team beamlines, which are funded by the
  NIGMS (P30 GM124165). The research used resources of the Advanced Photon Source,
  a US DoE Office of Science User Facility operated for the DoE Office of Science
  by Argonne National Laboratory under contract DE-AC02-06CH11357. The Berkeley Center
  for Structural Biology is supported by the NIH, NIGMS and the Howard Hughes Medical
  Institute. The Advanced Light Source is supported by the Director, Office of Science,
  Office of Basic Energy Sciences and US DoE (DE-AC02-05CH11231).
article_processing_charge: Yes (in subscription journal)
article_type: original
author:
- first_name: Adam J.
  full_name: Broerman, Adam J.
  last_name: Broerman
- first_name: Christoph
  full_name: Pollmann, Christoph
  last_name: Pollmann
- first_name: Yang
  full_name: Zhao, Yang
  last_name: Zhao
- first_name: Mauriz A.
  full_name: Lichtenstein, Mauriz A.
  last_name: Lichtenstein
- first_name: Mark D.
  full_name: Jackson, Mark D.
  last_name: Jackson
- first_name: Maxx H.
  full_name: Tessmer, Maxx H.
  last_name: Tessmer
- first_name: Won Hee
  full_name: Ryu, Won Hee
  last_name: Ryu
- first_name: Masato
  full_name: Ogishi, Masato
  last_name: Ogishi
- first_name: Mohamad H.
  full_name: Abedi, Mohamad H.
  last_name: Abedi
- first_name: Danny D.
  full_name: Sahtoe, Danny D.
  last_name: Sahtoe
- first_name: Aza
  full_name: Allen, Aza
  last_name: Allen
- first_name: Alex
  full_name: Kang, Alex
  last_name: Kang
- first_name: Joshmyn
  full_name: De La Cruz, Joshmyn
  last_name: De La Cruz
- first_name: Evans
  full_name: Brackenbrough, Evans
  last_name: Brackenbrough
- first_name: Banumathi
  full_name: Sankaran, Banumathi
  last_name: Sankaran
- first_name: Asim K.
  full_name: Bera, Asim K.
  last_name: Bera
- first_name: Daniel M.
  full_name: Zuckerman, Daniel M.
  last_name: Zuckerman
- first_name: Stefan
  full_name: Stoll, Stefan
  last_name: Stoll
- first_name: K. Christopher
  full_name: Garcia, K. Christopher
  last_name: Garcia
- first_name: Florian M
  full_name: Praetorius, Florian M
  id: dfec9381-4341-11ee-8fd8-faa02bba7d62
  last_name: Praetorius
  orcid: 0000-0002-0806-8101
- first_name: Jacob
  full_name: Piehler, Jacob
  last_name: Piehler
- first_name: David
  full_name: Baker, David
  last_name: Baker
citation:
  ama: Broerman AJ, Pollmann C, Zhao Y, et al. Design of facilitated dissociation
    enables timing of cytokine signalling. <i>Nature</i>. 2025;647:528-535. doi:<a
    href="https://doi.org/10.1038/s41586-025-09549-z">10.1038/s41586-025-09549-z</a>
  apa: Broerman, A. J., Pollmann, C., Zhao, Y., Lichtenstein, M. A., Jackson, M. D.,
    Tessmer, M. H., … Baker, D. (2025). Design of facilitated dissociation enables
    timing of cytokine signalling. <i>Nature</i>. Springer Nature. <a href="https://doi.org/10.1038/s41586-025-09549-z">https://doi.org/10.1038/s41586-025-09549-z</a>
  chicago: Broerman, Adam J., Christoph Pollmann, Yang Zhao, Mauriz A. Lichtenstein,
    Mark D. Jackson, Maxx H. Tessmer, Won Hee Ryu, et al. “Design of Facilitated Dissociation
    Enables Timing of Cytokine Signalling.” <i>Nature</i>. Springer Nature, 2025.
    <a href="https://doi.org/10.1038/s41586-025-09549-z">https://doi.org/10.1038/s41586-025-09549-z</a>.
  ieee: A. J. Broerman <i>et al.</i>, “Design of facilitated dissociation enables
    timing of cytokine signalling,” <i>Nature</i>, vol. 647. Springer Nature, pp.
    528–535, 2025.
  ista: Broerman AJ, Pollmann C, Zhao Y, Lichtenstein MA, Jackson MD, Tessmer MH,
    Ryu WH, Ogishi M, Abedi MH, Sahtoe DD, Allen A, Kang A, De La Cruz J, Brackenbrough
    E, Sankaran B, Bera AK, Zuckerman DM, Stoll S, Garcia KC, Praetorius FM, Piehler
    J, Baker D. 2025. Design of facilitated dissociation enables timing of cytokine
    signalling. Nature. 647, 528–535.
  mla: Broerman, Adam J., et al. “Design of Facilitated Dissociation Enables Timing
    of Cytokine Signalling.” <i>Nature</i>, vol. 647, Springer Nature, 2025, pp. 528–35,
    doi:<a href="https://doi.org/10.1038/s41586-025-09549-z">10.1038/s41586-025-09549-z</a>.
  short: A.J. Broerman, C. Pollmann, Y. Zhao, M.A. Lichtenstein, M.D. Jackson, M.H.
    Tessmer, W.H. Ryu, M. Ogishi, M.H. Abedi, D.D. Sahtoe, A. Allen, A. Kang, J. De
    La Cruz, E. Brackenbrough, B. Sankaran, A.K. Bera, D.M. Zuckerman, S. Stoll, K.C.
    Garcia, F.M. Praetorius, J. Piehler, D. Baker, Nature 647 (2025) 528–535.
corr_author: '1'
date_created: 2025-10-05T22:01:36Z
date_published: 2025-11-13T00:00:00Z
date_updated: 2026-01-05T13:18:17Z
day: '13'
ddc:
- '570'
department:
- _id: FlPr
doi: 10.1038/s41586-025-09549-z
external_id:
  isi:
  - '001577755600001'
  pmid:
  - '40993395'
file:
- access_level: open_access
  checksum: b4ec44134e2eb320a724dc29158dfda2
  content_type: application/pdf
  creator: dernst
  date_created: 2026-01-05T13:17:47Z
  date_updated: 2026-01-05T13:17:47Z
  file_id: '20951'
  file_name: 2025_Nature_Broerman.pdf
  file_size: 22099921
  relation: main_file
  success: 1
file_date_updated: 2026-01-05T13:17:47Z
has_accepted_license: '1'
intvolume: '       647'
isi: 1
language:
- iso: eng
month: '11'
oa: 1
oa_version: Published Version
page: 528-535
pmid: 1
publication: Nature
publication_identifier:
  eissn:
  - 1476-4687
  issn:
  - 0028-0836
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: Design of facilitated dissociation enables timing of cytokine signalling
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: 647
year: '2025'
...
---
DOAJ_listed: '1'
OA_place: publisher
OA_type: gold
_id: '18757'
abstract:
- lang: eng
  text: 'Segmentation is a critical data processing step in many applications of cryo-electron
    tomography. Downstream analyses, such as subtomogram averaging, are often based
    on segmentation results, and are thus critically dependent on the availability
    of open-source software for accurate as well as high-throughput tomogram segmentation.
    There is a need for more user-friendly, flexible, and comprehensive segmentation
    software that offers an insightful overview of all steps involved in preparing
    automated segmentations. Here, we present Ais: a dedicated tomogram segmentation
    package that is geared towards both high performance and accessibility, available
    on GitHub. In this report, we demonstrate two common processing steps that can
    be greatly accelerated with Ais: particle picking for subtomogram averaging, and
    generating many-feature segmentations of cellular architecture based on in situ
    tomography data. Featuring comprehensive annotation, segmentation, and rendering
    functionality, as well as an open repository for trained models at aiscryoet.org,
    we hope that Ais will help accelerate research and dissemination of data involving
    cryoET.'
acknowledgement: 'We thank A Koster and M Barcena for helpful discussions and kindly
  sharing the coronaviral replication organelle datasets. We are also grateful to
  van den Hoek et al., 2022 and Wu et al., 2023, for uploading the data that we used
  for Figure 5 onto EMPIAR and EMDB, as well as to the authors of various other datasets
  uploaded to these databases that are not discussed in this manuscript but that were
  useful for testing the software. We also thank the reviewers, whose comments were
  very helpful in improving the manuscript and the software. Finally, we are grateful
  the early Ais users who provided us with feedback on the software and reported issues.
  This research was supported by the following grants to THS: European Research Council
  H202 Grant 759517; European Union’s Horizon Europe Program IMAGINE grant 101094250,
  and the Netherlands Organization for Scientific Research Grant VI.Vidi.193.014.'
article_number: '98552'
article_processing_charge: Yes
article_type: original
author:
- first_name: Mart G.F.
  full_name: Last, Mart G.F.
  last_name: Last
- first_name: Leoni
  full_name: Abendstein, Leoni
  id: 14f1f051-cd9d-11ef-9c94-8b942a882560
  last_name: Abendstein
  orcid: 0000-0001-7634-5353
- first_name: Lenard M.
  full_name: Voortman, Lenard M.
  last_name: Voortman
- first_name: Thomas H.
  full_name: Sharp, Thomas H.
  last_name: Sharp
citation:
  ama: Last MGF, Abendstein L, Voortman LM, Sharp TH. Streamlining segmentation of
    cryo-electron tomography datasets with Ais. <i>eLife</i>. 2024;13. doi:<a href="https://doi.org/10.7554/eLife.98552">10.7554/eLife.98552</a>
  apa: Last, M. G. F., Abendstein, L., Voortman, L. M., &#38; Sharp, T. H. (2024).
    Streamlining segmentation of cryo-electron tomography datasets with Ais. <i>ELife</i>.
    eLife Sciences Publications. <a href="https://doi.org/10.7554/eLife.98552">https://doi.org/10.7554/eLife.98552</a>
  chicago: Last, Mart G.F., Leoni Abendstein, Lenard M. Voortman, and Thomas H. Sharp.
    “Streamlining Segmentation of Cryo-Electron Tomography Datasets with Ais.” <i>ELife</i>.
    eLife Sciences Publications, 2024. <a href="https://doi.org/10.7554/eLife.98552">https://doi.org/10.7554/eLife.98552</a>.
  ieee: M. G. F. Last, L. Abendstein, L. M. Voortman, and T. H. Sharp, “Streamlining
    segmentation of cryo-electron tomography datasets with Ais,” <i>eLife</i>, vol.
    13. eLife Sciences Publications, 2024.
  ista: Last MGF, Abendstein L, Voortman LM, Sharp TH. 2024. Streamlining segmentation
    of cryo-electron tomography datasets with Ais. eLife. 13, 98552.
  mla: Last, Mart G. F., et al. “Streamlining Segmentation of Cryo-Electron Tomography
    Datasets with Ais.” <i>ELife</i>, vol. 13, 98552, eLife Sciences Publications,
    2024, doi:<a href="https://doi.org/10.7554/eLife.98552">10.7554/eLife.98552</a>.
  short: M.G.F. Last, L. Abendstein, L.M. Voortman, T.H. Sharp, ELife 13 (2024).
date_created: 2025-01-05T23:01:57Z
date_published: 2024-12-20T00:00:00Z
date_updated: 2025-01-08T08:52:51Z
day: '20'
ddc:
- '570'
department:
- _id: FlPr
doi: 10.7554/eLife.98552
external_id:
  pmid:
  - '39704648'
file:
- access_level: open_access
  checksum: a4f0f906e4d5c1078208b317e78699d1
  content_type: application/pdf
  creator: dernst
  date_created: 2025-01-08T08:51:45Z
  date_updated: 2025-01-08T08:51:45Z
  file_id: '18774'
  file_name: 2024_eLife_Last.pdf
  file_size: 7445664
  relation: main_file
  success: 1
file_date_updated: 2025-01-08T08:51:45Z
has_accepted_license: '1'
intvolume: '        13'
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
pmid: 1
publication: eLife
publication_identifier:
  eissn:
  - 2050-084X
publication_status: published
publisher: eLife Sciences Publications
quality_controlled: '1'
scopus_import: '1'
status: public
title: Streamlining segmentation of cryo-electron tomography datasets with Ais
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: '2024'
...
---
_id: '17463'
abstract:
- lang: eng
  text: Allosteric modulation of protein function, wherein the binding of an effector
    to a protein triggers conformational changes at distant functional sites, plays
    a central part in the control of metabolism and cell signalling1,2,3. There has
    been considerable interest in designing allosteric systems, both to gain insight
    into the mechanisms underlying such ‘action at a distance’ modulation and to create
    synthetic proteins whose functions can be regulated by effectors4,5,6,7. However,
    emulating the subtle conformational changes distributed across many residues,
    characteristic of natural allosteric proteins, is a significant challenge8,9.
    Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity10, we
    investigate the de novo design of allostery through rigid-body coupling of peptide-switchable
    hinge modules11 to protein interfaces12 that direct the formation of alternative
    oligomeric states. We find that this approach can be used to generate a wide variety
    of allosterically switchable systems, including cyclic rings that incorporate
    or eject subunits in response to peptide binding and dihedral cages that undergo
    effector-induced disassembly. Size-exclusion chromatography, mass photometry13
    and electron microscopy reveal that these designed allosteric protein assemblies
    closely resemble the design models in both the presence and absence of peptide
    effectors and can have ligand-binding cooperativity comparable to classic natural
    systems such as haemoglobin14. Our results indicate that allostery can arise from
    global coupling of the energetics of protein substructures without optimized side-chain–side-chain
    allosteric communication pathways and provide a roadmap for generating allosterically
    triggerable delivery systems, protein nanomachines and cellular feedback control
    circuitry.
acknowledgement: We thank D. D. Sahtoe, R. D. Kiber, Y. Hsia, N. Bethel and A. Favor
  for helpful discussions and K. VanWormer and L. Goldschmidt for technical support.
  We also thank X. Li and M. Lamb for mass spectrometry support. This work was supported
  by the Washington Research Foundation Postdoctoral Fellowship (grant no. GR027504,
  A. Pillai), a National Science Foundation Graduate Research Fellowship (grant no.
  DGE-2140004, A.I.), a Human Frontier Science Program Long Term Fellowship (grant
  no. LT000880/2019, F.P.), the Audacious Project at the Institute for Protein Design
  (A.B., A. Pillai, A. Philomin, A.I. and D.B.), a National Energy Research Scientific
  Computing Centre award (grant no. BER-ERCAP0022018), the Howard Hughes Medical Institute
  (D.B.), the Open Philanthropy Project Improving Protein Design Fund (P.J.Y.L., C.D.
  and D.B.) a gift from Microsoft (D.B.) and a grant from DARPA supporting the Harnessing
  Enzymatic Activity for Lifesaving Remedies programme (grant no. HR001120S0052, contract
  no. HR0011-21-2-0012, D.B.).
article_processing_charge: Yes (in subscription journal)
article_type: original
author:
- first_name: Arvind
  full_name: Pillai, Arvind
  last_name: Pillai
- first_name: Abbas
  full_name: Idris, Abbas
  last_name: Idris
- first_name: Annika
  full_name: Philomin, Annika
  last_name: Philomin
- first_name: Connor
  full_name: Weidle, Connor
  last_name: Weidle
- first_name: Rebecca
  full_name: Skotheim, Rebecca
  last_name: Skotheim
- first_name: Philip J.Y.
  full_name: Leung, Philip J.Y.
  last_name: Leung
- first_name: Adam
  full_name: Broerman, Adam
  last_name: Broerman
- first_name: Cullen
  full_name: Demakis, Cullen
  last_name: Demakis
- first_name: Andrew J.
  full_name: Borst, Andrew J.
  last_name: Borst
- first_name: Florian M
  full_name: Praetorius, Florian M
  id: dfec9381-4341-11ee-8fd8-faa02bba7d62
  last_name: Praetorius
- first_name: David
  full_name: Baker, David
  last_name: Baker
citation:
  ama: Pillai A, Idris A, Philomin A, et al. De novo design of allosterically switchable
    protein assemblies. <i>Nature</i>. 2024;632:911–920. doi:<a href="https://doi.org/10.1038/s41586-024-07813-2">10.1038/s41586-024-07813-2</a>
  apa: Pillai, A., Idris, A., Philomin, A., Weidle, C., Skotheim, R., Leung, P. J.
    Y., … Baker, D. (2024). De novo design of allosterically switchable protein assemblies.
    <i>Nature</i>. Springer Nature. <a href="https://doi.org/10.1038/s41586-024-07813-2">https://doi.org/10.1038/s41586-024-07813-2</a>
  chicago: Pillai, Arvind, Abbas Idris, Annika Philomin, Connor Weidle, Rebecca Skotheim,
    Philip J.Y. Leung, Adam Broerman, et al. “De Novo Design of Allosterically Switchable
    Protein Assemblies.” <i>Nature</i>. Springer Nature, 2024. <a href="https://doi.org/10.1038/s41586-024-07813-2">https://doi.org/10.1038/s41586-024-07813-2</a>.
  ieee: A. Pillai <i>et al.</i>, “De novo design of allosterically switchable protein
    assemblies,” <i>Nature</i>, vol. 632. Springer Nature, pp. 911–920, 2024.
  ista: Pillai A, Idris A, Philomin A, Weidle C, Skotheim R, Leung PJY, Broerman A,
    Demakis C, Borst AJ, Praetorius FM, Baker D. 2024. De novo design of allosterically
    switchable protein assemblies. Nature. 632, 911–920.
  mla: Pillai, Arvind, et al. “De Novo Design of Allosterically Switchable Protein
    Assemblies.” <i>Nature</i>, vol. 632, Springer Nature, 2024, pp. 911–920, doi:<a
    href="https://doi.org/10.1038/s41586-024-07813-2">10.1038/s41586-024-07813-2</a>.
  short: A. Pillai, A. Idris, A. Philomin, C. Weidle, R. Skotheim, P.J.Y. Leung, A.
    Broerman, C. Demakis, A.J. Borst, F.M. Praetorius, D. Baker, Nature 632 (2024)
    911–920.
corr_author: '1'
date_created: 2024-08-25T22:01:08Z
date_published: 2024-08-22T00:00:00Z
date_updated: 2025-09-08T09:00:16Z
day: '22'
ddc:
- '570'
department:
- _id: FlPr
doi: 10.1038/s41586-024-07813-2
external_id:
  isi:
  - '001300534300019'
  pmid:
  - '39143214'
file:
- access_level: open_access
  checksum: 39127601621a360ec0edc538627eb211
  content_type: application/pdf
  creator: dernst
  date_created: 2024-09-09T12:01:14Z
  date_updated: 2024-09-09T12:01:14Z
  file_id: '18005'
  file_name: 2024_Nature_Pillai.pdf
  file_size: 16572040
  relation: main_file
  success: 1
file_date_updated: 2024-09-09T12:01:14Z
has_accepted_license: '1'
intvolume: '       632'
isi: 1
language:
- iso: eng
month: '08'
oa: 1
oa_version: Published Version
page: '911–920 '
pmid: 1
publication: Nature
publication_identifier:
  eissn:
  - 1476-4687
  issn:
  - 0028-0836
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: De novo design of allosterically switchable protein assemblies
tmp:
  image: /images/cc_by_nc_nd.png
  legal_code_url: https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode
  name: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
    (CC BY-NC-ND 4.0)
  short: CC BY-NC-ND (4.0)
type: journal_article
user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345
volume: 632
year: '2024'
...