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- EMDB-38112: local refinement of MCM_apo -

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Basic information

Entry
Database: EMDB / ID: EMD-38112
Titlelocal refinement of MCM_apo
Map datalocal refinement of MCM_apo
Sample
  • Complex: MCM homohexamer
KeywordsHelicase / Replication / HYDROLASE
Biological speciesThermococcus kodakarensis (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsMa J / Yi G / Ye M / MacGregor-Chatwin C / Sheng Y / Lu Y / Li M / Gilbert RJC / Zhang P
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM.
Authors: Jianbing Ma / Gangshun Yi / Mingda Ye / Craig MacGregor-Chatwin / Yuewen Sheng / Ying Lu / Ming Li / Qingrong Li / Dong Wang / Robert J C Gilbert / Peijun Zhang /
Abstract: The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging ...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.
History
DepositionNov 25, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38112.png

Downloads & links

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Map

FileDownload / File: emd_38112.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal refinement of MCM_apo
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 411.648 Å		1.07 Å/pix.
x 384 pix.
= 411.648 Å		1.07 Å/pix.
x 384 pix.
= 411.648 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.072 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-1.0349288 - 1.6248213
Average (Standard dev.)0.00023787991 (±0.013664109)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 411.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A of local refinement of MCM apo

Fileemd_38112_half_map_1.map
Annotationhalf map A of local refinement of MCM_apo
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B of local refinement of MCM apo

Fileemd_38112_half_map_2.map
Annotationhalf map B of local refinement of MCM_apo
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MCM homohexamer

EntireName: MCM homohexamer
Components
  • Complex: MCM homohexamer

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Supramolecule #1: MCM homohexamer

SupramoleculeName: MCM homohexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermococcus kodakarensis (archaea)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: Generated using ab-initio reconstruction routine in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 445096
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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