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Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM

Jianbing Ma, Gangshun Yi, Mingda Ye, Craig MacGregor-Chatwin, Yuewen Sheng, Ying Lu, Ming Li, Qingrong Li, Dong Wang, Robert J. C. Gilbert and Peijun Zhang (peijun.zhang@strubi.ox.ac.uk)
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Jianbing Ma: University of Oxford
Gangshun Yi: University of Oxford
Mingda Ye: University of Oxford
Craig MacGregor-Chatwin: Harwell Science and Innovation Campus
Yuewen Sheng: Harwell Science and Innovation Campus
Ying Lu: Chinese Academy of Sciences
Ming Li: Chinese Academy of Sciences
Qingrong Li: University of California San Diego
Dong Wang: University of California San Diego
Robert J. C. Gilbert: University of Oxford
Peijun Zhang: University of Oxford

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract 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.

Date: 2024
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DOI: 10.1038/s41467-024-53745-w

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