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- EMDB-6535: Structure of the eukaryotic replicative CMG helicase and pumpjack... -

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Basic information

Entry
Database: EMDB / ID: EMD-6535
TitleStructure of the eukaryotic replicative CMG helicase and pumpjack motion
Map dataStructure of CMG with "Flat" Mcm2-7 ring
Sample
  • Sample: Saccharomyces cerevisiae CMG complex
  • Protein or peptide: CMG complex
KeywordsCMG helicase / Cryo-EM
Function / homology
Function and homology information


Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / premeiotic DNA replication ...Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / : / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / nuclear replication fork / : / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / DNA-templated DNA replication / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
: / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily ...: / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / : / MCM3 winged helix domain / MCM4, winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsYuan Z / Bai L / Sun J / Georgescu RE / Liu J / O'Donnell ME / Li H
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation.
Authors: Zuanning Yuan / Lin Bai / Jingchuan Sun / Roxana Georgescu / Jun Liu / Michael E O'Donnell / Huilin Li /
Abstract: The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals ...The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.
History
DepositionNov 23, 2015-
Header (metadata) releaseFeb 10, 2016-
Map releaseFeb 10, 2016-
UpdateApr 27, 2016-
Current statusApr 27, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jc5
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6535.jpg

Downloads & links

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Map

FileDownload / File: emd_6535.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of CMG with "Flat" Mcm2-7 ring
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 258.56 Å		1.01 Å/pix.
x 256 pix.
= 258.56 Å		1.01 Å/pix.
x 256 pix.
= 258.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.018 / Movie #1: 0.015
Minimum - Maximum-0.02877966 - 0.06427838
Average (Standard dev.)0.00066107 (±0.00432134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.011.011.01
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z258.560258.560258.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0290.0640.001

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae CMG complex

EntireName: Saccharomyces cerevisiae CMG complex
Components
  • Sample: Saccharomyces cerevisiae CMG complex
  • Protein or peptide: CMG complex

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Supramolecule #1000: Saccharomyces cerevisiae CMG complex

SupramoleculeName: Saccharomyces cerevisiae CMG complex / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: CMG complex

MacromoleculeName: CMG complex / type: protein_or_peptide / ID: 1 / Name.synonym: CMG / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration0.6 mg/mL
BufferDetails: 20 mM Tris acetate, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
GridDetails: 400 mesh holey carbon C-flat grid, glow-discharged in air
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateAug 1, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 8000 / Average electron dose: 50 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49505 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAll steps, including automatic particle picking, 2D classification, 3D classification, and 3D refinement were performed in Relion 1.4.
CTF correctionDetails: CTFFIND4
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: OTHER / Software - Name: Relion_1.4 / Number images used: 178530

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Atomic model buiding 1

Initial modelPDB ID:

2q9q

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jc5:
Structure of the eukaryotic replicative CMG helicase and pumpjack motion

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