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- EMDB-10511: Conformation1_Mcm4,7_MultiBody -

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Basic information

Entry
Database: EMDB / ID: EMD-10511
TitleConformation1_Mcm4,7_MultiBody
Map dataConformation1_Mcm4,7_MultiBody
Sample
  • Complex: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBaretic D / Jenkyn-Bedford M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/12 United Kingdom
CitationJournal: Mol Cell / Year: 2020
Title: Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.
Authors: Domagoj Baretić / Michael Jenkyn-Bedford / Valentina Aria / Giuseppe Cannone / Mark Skehel / Joseph T P Yeeles /
Abstract: The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric ...The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.
History
DepositionNov 21, 2019-
Header (metadata) releaseMay 6, 2020-
Map releaseMay 6, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10511.png

Downloads & links

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Map

FileDownload / File: emd_10511.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConformation1_Mcm4,7_MultiBody
Voxel sizeX=Y=Z: 1.049 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.024765363 - 0.05954113
Average (Standard dev.)-0.00012026888 (±0.001186623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0491.0491.049
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z377.640377.640377.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0250.060-0.000

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Supplemental data

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Sample components

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Entire : Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

EntireName: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
Components
  • Complex: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

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Supramolecule #1: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

SupramoleculeName: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#16
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.4 MDa

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaOAcsodium acetate
0.5 mMTCEPtris(2-carboxyethyl)phosphine
0.1 mMAMP-PNPadenylyl-imidodiphosphate
0.7 mMMg(OAc)2magnesium acetate
0.005 %TWEEN20Polyoxyethylene (20) sorbitan monolaurate
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid ethane..
DetailsIn vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork

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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 K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 2 / Number real images: 6682 / Average exposure time: 7.0 sec. / Average electron dose: 37.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 130000
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

Particle selectionNumber selected: 632000
CTF correctionSoftware - Name: Gctf (ver. 0.50)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5u8s

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Details: Multi-Body refinement / Number images used: 198000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.6)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5u8s

DetailsISOLDE version 1.0b3 was also used for atomic coordinate refinement
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 20 / Target criteria: FSC 0.5

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