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

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

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Abstract The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous chervo jacke herren 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 here 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.