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

DOI: 10.1038/s41467-024-53745-w

Authors: Jianbing Ma (Wellcome Centre for Human Genetics, University of Oxford; Institute of Biophysics, Chinese Academy of Science; Institute of Physics, Chinese Academy of Sciences) , Gangshun Yi (Wellcome Centre for Human Genetics, University of Oxford; Diamond Light Source; Magdalen College, University of Oxford) , Mingda Ye (University of Oxford) , Craig Macgregor-Chatwin (Diamond Light Source) , Yuewen Sheng (Diamond Light Source) , Ying Lu (Institute of Physics, Chinese Academy of Sciences) , Ming Li (Institute of Physics, Chinese Academy of Sciences) , Qingrong Li (University of California San Diego) , Dong Wang (University of California San Diego) , Robert J. C. Gilbert (Wellcome Centre for Human Genetics, University of Oxford; Magdalen College, University of Oxford) , Peijun Zhang (Wellcome Centre for Human Genetics, University of Oxford; Diamond Light Source; Chinese Academy of Medical Sciences Oxford Institute, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Communications , VOL 15

State: Published (Approved)
Published: December 2024
Diamond Proposal Number(s): 29812

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.

Subject Areas: Biology and Bio-materials, Technique Development

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond , Krios II-Titan Krios II at Diamond , Krios IV-Titan Krios IV at Diamond

Added On: 02/12/2024 09:11

Documents:
s41467-024-53745-w.pdf

Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

Technical Tags:

Microscopy Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)