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 majo...
| Hauptverfasser: | , , , , , , , , , , |
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| Format: | Journal article |
| Sprache: | English |
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Nature Research
2024
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| _version_ | 1826316227028451328 |
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| author | Ma, J Yi, G Ye, M MacGregor-Chatwin, C Sheng, Y Lu, Y Li, M Li, Q Wang, D Gilbert, RJC Zhang, P |
| author_facet | Ma, J Yi, G Ye, M MacGregor-Chatwin, C Sheng, Y Lu, Y Li, M Li, Q Wang, D Gilbert, RJC Zhang, P |
| author_sort | Ma, J |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-12-09T03:39:44Z |
| format | Journal article |
| id | oxford-uuid:b7f7f2e8-c034-47e0-a9b6-a7ad4b020850 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:39:44Z |
| publishDate | 2024 |
| publisher | Nature Research |
| record_format | dspace |
| spelling | oxford-uuid:b7f7f2e8-c034-47e0-a9b6-a7ad4b0208502024-12-06T20:04:16ZOpen architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEMJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:b7f7f2e8-c034-47e0-a9b6-a7ad4b020850EnglishJisc Publications RouterNature Research2024Ma, JYi, GYe, MMacGregor-Chatwin, CSheng, YLu, YLi, MLi, QWang, DGilbert, RJCZhang, PThe 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. |
| spellingShingle | Ma, J Yi, G Ye, M MacGregor-Chatwin, C Sheng, Y Lu, Y Li, M Li, Q Wang, D Gilbert, RJC Zhang, P Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title | Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title_full | Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title_fullStr | Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title_full_unstemmed | Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title_short | Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM |
| title_sort | open architecture of archaea mcm and dsdna complexes resolved using monodispersed streptavidin affinity cryoem |
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