Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM
The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.
Authors
- Robert J.C. Gilbert (ORCID: https://orcid.org/0000-0001-9336-5604)
- Craig MacGregor-Chatwin (ORCID: https://orcid.org/0000-0002-3995-3373)
- Gangshun Yi (ORCID: https://orcid.org/0009-0004-6509-4893)
- Mingda Ye (ORCID: https://orcid.org/0000-0001-6324-4238)
- Qingrong Li (ORCID: https://orcid.org/0000-0002-8583-3620)
- Jianbing Ma (ORCID: https://orcid.org/0000-0002-1665-1428)
- Ying Lü (ORCID: https://orcid.org/0000-0002-8421-7228)
- Yuewen Sheng (ORCID: https://orcid.org/0000-0003-3067-9520)
- Ming Li (ORCID: https://orcid.org/0000-0002-5328-5826)
- Dong Wang (ORCID: https://orcid.org/0000-0002-2829-1546)
- Peijun Zhang (ORCID: https://orcid.org/0000-0003-1803-691X)
Institutions
- Centre for Human Genetics (GB)
- Chinese Academy of Sciences (CN)
- Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
- Diamond Light Source (GB)
- University of California San Diego (US)
- University of Oxford (GB)
- Institute of Biophysics (CN)
- Institute of Physics (CN)
- National Laboratory for Superconductivity (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2024-11-27
- DOI
- https://doi.org/10.1038/s41467-024-53745-w
- Citations
- 4
- Primary Topic
- Bacteriophages and microbial interactions
- Type
- article
- Field-Weighted Citation Impact
- 1.84
Funders
- Wellcome Trust
- National Institute for Health and Care Research
- National Natural Science Foundation of China
- National Institutes of Health
- Directorate for Biological Sciences
- Biotechnology and Biological Sciences Research Council
- National Key Research and Development Program of China
- Biomedical Research Council
- NIHR Oxford Biomedical Research Centre