Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation.
| Title: | Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation. |
|---|---|
| Authors: | Cookis T; Department of Molecular and Cell Biology, University of California, Berkeley; trinity_cookis@berkeley.edu.; Sauer P; California Institute for Quantitative Biology (QB3), University of California, Berkeley; Howard Hughes Medical Institute, University of California, Berkeley.; Poepsel S; Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne; Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne.; Han BG; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory.; Herbst DA; Department of Molecular and Cell Biology, University of California, Berkeley; California Institute for Quantitative Biology (QB3), University of California, Berkeley; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory.; Glaeser R; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory.; Nogales E; Department of Molecular and Cell Biology, University of California, Berkeley; California Institute for Quantitative Biology (QB3), University of California, Berkeley; Howard Hughes Medical Institute, University of California, Berkeley; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory. |
| Source: | Journal of visualized experiments : JoVE [J Vis Exp] 2023 Dec 29 (202). Date of Electronic Publication: 2023 Dec 29. |
| Publication Type: | Journal Article; Video-Audio Media; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
| Language: | English |
| Journal Info: | Publisher: MYJoVE Corporation Country of Publication: United States NLM ID: 101313252 Publication Model: Electronic Cited Medium: Internet ISSN: 1940-087X (Electronic) Linking ISSN: 1940087X NLM ISO Abbreviation: J Vis Exp Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: [Boston, Mass. : MYJoVE Corporation, 2006]- |
| MeSH Terms: | Carbon*/chemistry ; Biotin*; Cryoelectron Microscopy/methods ; Streptavidin/chemistry ; Water |
| Abstract: | Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community. |
| References: | Proc Natl Acad Sci U S A. 2020 Sep 29;117(39):24269-24273. (PMID: 32913054); J Struct Biol. 2014 Jan;185(1):42-7. (PMID: 24269484); J Struct Biol. 2010 Apr;170(1):152-6. (PMID: 20035878); J Microsc. 2019 Oct;276(1):39-45. (PMID: 31553060); J Struct Biol. 2016 Aug;195(2):238-244. (PMID: 27320699); Elife. 2018 May 29;7:. (PMID: 29809143); Science. 1972 Mar 3;175(4025):1000-1. (PMID: 4333169); Science. 2021 Jan 22;371(6527):. (PMID: 33479123); J Struct Biol. 2019 Sep 1;207(3):270-278. (PMID: 31200019); Nat Methods. 2017 Apr;14(4):331-332. (PMID: 28250466); J Struct Biol. 2012 Oct;180(1):249-53. (PMID: 22584152); Acta Crystallogr D Struct Biol. 2018 Jun 1;74(Pt 6):560-571. (PMID: 29872006); Nature. 2022 Sep;609(7928):835-845. (PMID: 36045294) |
| Grant Information: | R21 GM135666 United States GM NIGMS NIH HHS; R35 GM127018 United States GM NIGMS NIH HHS; T32 GM008295 United States GM NIGMS NIH HHS; United States HHMI Howard Hughes Medical Institute |
| Substance Nomenclature: | 9013-20-1 (Streptavidin); 7440-44-0 (Carbon); 6SO6U10H04 (Biotin); 059QF0KO0R (Water) |
| Entry Date(s): | Date Created: 20240115 Date Completed: 20240116 Latest Revision: 20250219 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC11293040 |
| DOI: | 10.3791/66197 |
| PMID: | 38224121 |
| Database: | MEDLINE |
Journal Article; Video-Audio Media; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.