--- _id: '9205' abstract: - lang: eng text: Cryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins. acknowledged_ssus: - _id: EM-Fac acknowledgement: We thank the Electron Microscopy Facilities at the Institute of Science and Technology Austria and at the Vienna Biocenter for providing access and training for the electron microscopes. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 665385 . article_number: '102139' article_processing_charge: No article_type: original author: - first_name: Domen full_name: Kampjut, Domen id: 37233050-F248-11E8-B48F-1D18A9856A87 last_name: Kampjut - first_name: Julia full_name: Steiner, Julia id: 3BB67EB0-F248-11E8-B48F-1D18A9856A87 last_name: Steiner - 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, Steiner J, Sazanov LA. Cryo-EM grid optimization for membrane proteins. iScience. 2021;24(3). doi:10.1016/j.isci.2021.102139 apa: Kampjut, D., Steiner, J., & Sazanov, L. A. (2021). Cryo-EM grid optimization for membrane proteins. IScience. Elsevier. https://doi.org/10.1016/j.isci.2021.102139 chicago: Kampjut, Domen, Julia Steiner, and Leonid A Sazanov. “Cryo-EM Grid Optimization for Membrane Proteins.” IScience. Elsevier, 2021. https://doi.org/10.1016/j.isci.2021.102139. ieee: D. Kampjut, J. Steiner, and L. A. Sazanov, “Cryo-EM grid optimization for membrane proteins,” iScience, vol. 24, no. 3. Elsevier, 2021. ista: Kampjut D, Steiner J, Sazanov LA. 2021. Cryo-EM grid optimization for membrane proteins. iScience. 24(3), 102139. mla: Kampjut, Domen, et al. “Cryo-EM Grid Optimization for Membrane Proteins.” IScience, vol. 24, no. 3, 102139, Elsevier, 2021, doi:10.1016/j.isci.2021.102139. short: D. Kampjut, J. Steiner, L.A. Sazanov, IScience 24 (2021). date_created: 2021-02-28T23:01:24Z date_published: 2021-03-19T00:00:00Z date_updated: 2023-08-07T13:54:06Z day: '19' ddc: - '570' department: - _id: LeSa doi: 10.1016/j.isci.2021.102139 ec_funded: 1 external_id: isi: - '000631646000012' pmid: - '33665558' file: - access_level: open_access checksum: 50585447386fe5842f07ab9b3a66e7e9 content_type: application/pdf creator: dernst date_created: 2021-03-03T07:38:14Z date_updated: 2021-03-03T07:38:14Z file_id: '9219' file_name: 2021_iScience_Kampjut.pdf file_size: 7431411 relation: main_file success: 1 file_date_updated: 2021-03-03T07:38:14Z has_accepted_license: '1' intvolume: ' 24' isi: 1 issue: '3' language: - iso: eng month: '03' oa: 1 oa_version: Published Version pmid: 1 project: - _id: 2564DBCA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '665385' name: International IST Doctoral Program publication: iScience publication_identifier: eissn: - '25890042' publication_status: published publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: Cryo-EM grid optimization for membrane proteins 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: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 24 year: '2021' ...