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

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Basic information

Entry
Database: PDB / ID: 3jc5
TitleStructure of the eukaryotic replicative CMG helicase and pumpjack motion
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • Minichromosome maintenance protein 5
KeywordsHYDROLASE / CMG helicase / Cryo-EM
Function / homology
Function and homology information


Unwinding of DNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication ...Unwinding of DNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / 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 / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / DNA unwinding 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 / heterochromatin formation / DNA-templated DNA replication / 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
: / MCM3 winged helix domain / GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal ...: / MCM3 winged helix domain / GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / 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 / 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 / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM OB 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsLi, H. / Bai, L. / Yuan, Z. / Sun, J. / Georgescu, R.E. / Liu, J. / O'Donnell, M.E.
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 24, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 10, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 24, 2016Group: Database references
Revision 1.2Mar 16, 2016Group: Database references
Revision 1.3Jan 24, 2018Group: Data processing / Structure summary / Category: audit_author / em_software / Item: _audit_author.name / _em_software.name
Revision 1.4Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id

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Structure visualization

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
c: Cell division control protein 45
D: DNA replication complex GINS protein SLD5
B: DNA replication complex GINS protein PSF2
A: DNA replication complex GINS protein PSF1
C: DNA replication complex GINS protein PSF3


Theoretical massNumber of molelcules
Total (without water)785,75811
Polymers785,75811
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase

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Protein , 2 types, 2 molecules 5c

#4: Protein Minichromosome maintenance protein 5 / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, DNA helicase
#7: Protein Cell division control protein 45


Mass: 74173.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032

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DNA replication complex GINS protein ... , 4 types, 4 molecules DBAC

#8: Protein DNA replication complex GINS protein SLD5


Mass: 33983.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406
#9: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


Mass: 25096.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359
#10: Protein DNA replication complex GINS protein PSF1 / Partner of Sld five 1


Mass: 24157.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488
#11: Protein DNA replication complex GINS protein PSF3 / Partner of Sld five 3


Mass: 21977.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF3, YOL146W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12146

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Saccharomyces cerevisiae CMG complex / Type: COMPLEX
Molecular weightValue: 0.7 MDa / Experimental value: NO
Buffer solutionName: 20 mM Tris acetate, pH 7.5, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
pH: 7.5
Details: 20 mM Tris acetate, pH 7.5, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh holey carbon C-flat grid, glow-discharged in air
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %
Details: Blot for 3 seconds before plunging into liquid ethane (FEI VITROBOT MARK IV).
Method: Blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Aug 1, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Calibrated magnification: 49505 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansNum. digital images: 8000

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2RELION1.43D reconstruction
CTF correctionDetails: CTFFIND4
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Single particle reconstruction / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178530 / Nominal pixel size: 1.01 Å / Actual pixel size: 1.01 Å
Details: (Single particle details: All steps, including automatic particle picking, 2D classification, 3D classification, and 3D refinement were performed in Relion 1.4.) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 2Q9Q

2q9q

Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms40041 0 0 0 40041

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