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- PDB-6u0m: Structure of the S. cerevisiae replicative helicase CMG in comple... -

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Entry
Database: PDB / ID: 6u0m
TitleStructure of the S. cerevisiae replicative helicase CMG in complex with a forked DNA
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • DNA (15-MER)
  • DNA (26-MER)
  • Minichromosome maintenance protein 5
KeywordsREPLICATION/DNA / DNA replication / CMG-Mcm10 / REPLICATION-DNA complex
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 / GINS complex / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / 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 / GINS complex / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / premeiotic DNA replication / replication fork protection complex / 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 / DNA replication preinitiation complex / Activation of ATR in response to replication stress / MCM 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 / transcription elongation by RNA polymerase II / helicase activity / DNA-templated DNA replication / heterochromatin formation / single-stranded DNA binding / DNA helicase / DNA replication / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytoplasm
Similarity search - Function
Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #2050 / mini-chromosome maintenance (MCM) complex, chain A, domain 1 / mini-chromosome maintenance (MCM) complex, chain A, domain 1 / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / CDC45 family ...Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #2050 / mini-chromosome maintenance (MCM) complex, chain A, domain 1 / mini-chromosome maintenance (MCM) complex, chain A, domain 1 / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / : / MCM3 winged helix domain / GINS subunit, domain A / GINS complex protein helical bundle domain / GINS complex, subunit Psf1 / GINS, helical bundle-like domain superfamily / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / 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 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / P-loop containing nucleotide triphosphate hydrolases / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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 ...ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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: 3.9 Å
AuthorsYuan, Z. / Georgescu, R. / Bai, L. / Zhang, D. / O'Donnell, M. / Li, H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM111472 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)OD12272 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM45436 United States
CitationJournal: Nat Commun / Year: 2020
Title: DNA unwinding mechanism of a eukaryotic replicative CMG helicase.
Authors: Zuanning Yuan / Roxana Georgescu / Lin Bai / Dan Zhang / Huilin Li / Michael E O'Donnell /
Abstract: High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic ...High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.
History
DepositionAug 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
Revision 2.0Apr 22, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / database_PDB_caveat ...atom_site / database_PDB_caveat / em_software / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / ndb_struct_conf_na / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_entity_src_syn / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_prop / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_polymer_linkage / pdbx_validate_rmsd_angle / pdbx_validate_torsion / struct_conf / struct_conn / struct_conn_type / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range / struct_site_gen
Item: _em_software.category / _entity.formula_weight ..._em_software.category / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_ec / _entity.src_method / _entity_name_com.entity_id / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_poly.pdbx_strand_id / _entity_poly.type / _entity_src_gen.entity_id / _entity_src_gen.pdbx_end_seq_num / _pdbx_entity_src_syn.entity_id / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _pdbx_struct_assembly_prop.value / _pdbx_struct_sheet_hbond.range_1_label_asym_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_label_asym_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.pdbx_strand_id / _struct_ref_seq.seq_align_end / _struct_sheet_range.beg_label_asym_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_label_asym_id / _struct_sheet_range.end_label_seq_id / _struct_site_gen.label_asym_id
Description: Atomic clashes / Provider: author / Type: Coordinate replacement
Revision 2.1Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: DNA replication complex GINS protein PSF1
B: DNA replication complex GINS protein PSF2
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45
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
F: DNA (26-MER)
G: DNA (15-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)661,10116
Polymers659,58013
Non-polymers1,5223
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area69730 Å2
ΔGint-364 kcal/mol
Surface area264690 Å2

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Components

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

#1: Protein DNA replication complex GINS protein PSF1 / Partner of Sld five 1


Mass: 24230.576 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
#2: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


Mass: 23428.115 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
#3: Protein DNA replication complex GINS protein PSF3 / Partner of Sld five 3


Mass: 21659.705 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
#4: Protein DNA replication complex GINS protein SLD5


Mass: 33624.180 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

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Protein , 2 types, 2 molecules E5

#5: Protein Cell division control protein 45


Mass: 73810.250 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
#9: Protein Minichromosome maintenance protein 5 / Cell division control protein 46


Mass: 74401.297 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

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

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


Mass: 74987.070 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
#7: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 79986.094 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
#8: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 85821.812 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
#10: Protein DNA replication licensing factor MCM6


Mass: 73927.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091*PLUS
#11: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 82034.445 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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DNA chain , 2 types, 2 molecules FG

#12: DNA chain DNA (26-MER)


Mass: 7075.591 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#13: DNA chain DNA (15-MER)


Mass: 4593.011 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 1 types, 3 molecules

#14: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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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: CMG-forkDNA / Type: COMPLEX / Entity ID: #1-#13 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pRS
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162550 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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