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- EMDB-33320: Cryo-EM map of hMCM-DH R195A/L209G mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-33320
TitleCryo-EM map of hMCM-DH R195A/L209G mutant
Map data
Sample
  • Complex: humman MCM-DH
    • Protein or peptide: hMCM5
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsLi J / Dong JQ / Dang SY / Zhai YL
Funding support Hong Kong, 1 items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC) Hong Kong
CitationJournal: Cell / Year: 2023
Title: The human pre-replication complex is an open complex.
Authors: Jian Li / Jiangqing Dong / Weitao Wang / Daqi Yu / Xinyu Fan / Yan Chit Hui / Clare S K Lee / Wai Hei Lam / Nathan Alary / Yang Yang / Yingyi Zhang / Qian Zhao / Chun-Long Chen / Bik-Kwoon ...Authors: Jian Li / Jiangqing Dong / Weitao Wang / Daqi Yu / Xinyu Fan / Yan Chit Hui / Clare S K Lee / Wai Hei Lam / Nathan Alary / Yang Yang / Yingyi Zhang / Qian Zhao / Chun-Long Chen / Bik-Kwoon Tye / Shangyu Dang / Yuanliang Zhai /
Abstract: In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this ...In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.
History
DepositionApr 29, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33320.png

Downloads & links

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Map

FileDownload / File: emd_33320.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.1054487 - 1.9801762
Average (Standard dev.)-0.00035039464 (±0.041414008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #3

Fileemd_33320_additional_1.map
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Additional map: #2

Fileemd_33320_additional_2.map
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Additional map: #1

Fileemd_33320_additional_3.map
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Additional map: #4

Fileemd_33320_additional_4.map
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Half map: #2

Fileemd_33320_half_map_1.map
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Half map: #1

Fileemd_33320_half_map_2.map
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Sample components

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Entire : humman MCM-DH

EntireName: humman MCM-DH
Components
  • Complex: humman MCM-DH
    • Protein or peptide: hMCM5

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Supramolecule #1: humman MCM-DH

SupramoleculeName: humman MCM-DH / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: hMCM5

MacromoleculeName: hMCM5 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MSGFDDPGIF YSDSFGGDAQ ADEGQARKSQ LQRRFKEFLR QYRVGTDRTG FTFKYRDELK RHYNLGEYWI EVEMEDLASF DEDLADYLYK QPAEHLQLLE EAAKEVADEV TRPRPSGEEV LQDIQVMLKS DASPSSIRSL KSDMMSHLVK IPGIIIAASA VRAKATRISI ...String:
MSGFDDPGIF YSDSFGGDAQ ADEGQARKSQ LQRRFKEFLR QYRVGTDRTG FTFKYRDELK RHYNLGEYWI EVEMEDLASF DEDLADYLYK QPAEHLQLLE EAAKEVADEV TRPRPSGEEV LQDIQVMLKS DASPSSIRSL KSDMMSHLVK IPGIIIAASA VRAKATRISI QCRSCRNTLT NIAMRPGLEG YALPAKCNTD QAGRPKCPGD PYFIMPDKCK CVDFQTLKLQ ELPDAVPHGE MPRHMQLYCD RYLCDKVVPG NRVTIMGIYS IKKFGLTTSR GRDRVGVGIR SSYIRVLGIQ VDTDGSGRSF AGAVSPQEEE EFRRLAALPN VYEVISKSIA PSIFGGTDMK KAIACLLFGG SRKRLPDGLT RRGDINLLML GDPGTAKSQL LKFVEKCSPI GVYTSGKGSS AAGLTASVMR DPSSRNFIME GGAMVLADGG VVCIDEFDKM REDDRVAIHE AMEQQTISIA KAGITTTLNS RCSVLAAANS VFGRWDETKG EDNIDFMPTI LSRFDMIFIV KDEHNEERDV MLAKHVITLH VSALTQTQAV EGEIDLAKLK KFIAYCRVKC GPRLSAEAAE KLKNRYIIMR SGARQHERDS DRRSSIPITV RQLEAIVRIA EALSKMKLQP FATEADVEEA LRLFQVSTLD AALSGTLSGV EGFTSQEDQE MLSRIEKQLK RRFAIGSQVS EHSIIKDFTK QKYPEHAIHK VLQLMLRRGE IQHRMQRKVL YRLK

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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: 7.5
Component:
ConcentrationFormulaName
100.0 mML-GluL-Glutamic acid potassium salt
50.0 mMHEPESHEPES
1.0 mMEDTAEDTA
0.02 %NP-40NP-40
3.0 mMATPATP
2.0 mMNaFNaF
1.0 mMNa3VO4Na3VO4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 4748 / Average exposure time: 5.0 sec. / Average electron dose: 50.04 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.0.1) / Number images used: 349893
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.0.1)

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