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- PDB-6rqc: Cryo-EM structure of an MCM loading intermediate -

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Basic information

Entry
Database: PDB / ID: 6rqc
TitleCryo-EM structure of an MCM loading intermediate
Components
  • (DNA (88-MER)) x 2
  • (DNA replication licensing factor ...) x 5
  • (Origin recognition complex subunit ...) x 6
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / DNA Replication / Origin licensing / MCM2-7 helicase / Origin Recognition Complex
Function / homology
Function and homology information


CDC6 association with the ORC:origin complex / Assembly of the ORC complex at the origin of replication / Orc1 removal from chromatin / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding ...CDC6 association with the ORC:origin complex / Assembly of the ORC complex at the origin of replication / Orc1 removal from chromatin / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / premeiotic DNA replication / nuclear origin of replication recognition complex / 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 / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication checkpoint signaling / replication fork protection complex / mitotic DNA replication initiation / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / regulation of DNA replication / subtelomeric heterochromatin formation / DNA replication initiation / nucleosome binding / heterochromatin formation / DNA helicase activity / helicase activity / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / AAA domain / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal ...Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / AAA domain / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal / : / : / Origin recognition complex (ORC) subunit 4 C-terminus / Origin recognition complex (ORC) subunit 5 C-terminus / Origin recognition complex winged helix C-terminal / ORC5, lid domain / Orc1-like, AAA ATPase domain / AAA ATPase domain / Origin recognition complex subunit 2 / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / MCM4, winged helix 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 / Bromo adjacent homology (BAH) domain superfamily / Bromo adjacent homology domain / Bromo adjacent homology (BAH) domain / BAH domain / BAH domain profile. / 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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / EF-Hand 1, calcium-binding site / 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
ADENOSINE-5'-DIPHOSPHATE / 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 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 ...ADENOSINE-5'-DIPHOSPHATE / 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 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 6 / Origin recognition complex subunit 5 / DNA replication licensing factor MCM6 / Origin recognition complex subunit 1 / Origin recognition complex subunit 3 / Origin recognition complex subunit 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsMiller, T.C.R. / Locke, J. / Costa, A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
The Francis Crick InstituteFC0010065 United Kingdom
European Research Council820102 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM.
Authors: Thomas C R Miller / Julia Locke / Julia F Greiwe / John F X Diffley / Alessandro Costa /
Abstract: In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double- ...In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
History
DepositionMay 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 11, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Origin recognition complex subunit 1
B: Origin recognition complex subunit 2
C: Origin recognition complex subunit 3
D: Origin recognition complex subunit 4
E: Origin recognition complex subunit 5
F: Origin recognition complex subunit 6
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
X: DNA (88-MER)
Y: DNA (88-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,086,94329
Polymers1,083,31314
Non-polymers3,63015
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Origin recognition complex subunit ... , 6 types, 6 molecules ABCDEF

#1: Protein Origin recognition complex subunit 1 / / Origin recognition complex 120 kDa subunit


Mass: 108612.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Sequence includes an N-terminal CBP-tag and TEV cleavage site.
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC1, YML065W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54784
#2: Protein Origin recognition complex subunit 2 / / Origin recognition complex 71 kDa subunit


Mass: 71342.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC2, RRR1, SIR5, YBR060C, YBR0523 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P32833
#3: Protein Origin recognition complex subunit 3 / / Origin recognition complex 62 kDa subunit


Mass: 72161.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC3, OAF1, OIF1, YLL004W, L1365 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54790
#4: Protein Origin recognition complex subunit 4 / / Origin recognition complex 56 kDa subunit


Mass: 60772.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC4, YPR162C, P9325.5 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54791
#5: Protein Origin recognition complex subunit 5 / / Origin recognition complex 53 kDa subunit


Mass: 55347.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P50874
#6: Protein Origin recognition complex subunit 6 / / ACS-associated protein 1 / Origin recognition complex 50 kDa subunit


Mass: 50369.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: ORC6, AAP1, YHR118C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38826

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

#7: 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 S288c (yeast) / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29469, DNA helicase
#8: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 111720.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Sequence includes an N-terminal CBP-tag and TEV cleavage site.
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P24279, DNA helicase
#9: 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 S288c (yeast) / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P30665, DNA helicase
#11: 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 S288c (yeast) / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P53091, DNA helicase
#12: 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 S288c (yeast) / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38132, DNA helicase

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Protein , 1 types, 1 molecules 5

#10: 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 S288c (yeast) / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29496, DNA helicase

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DNA chain , 2 types, 2 molecules XY

#13: DNA chain DNA (88-MER)


Mass: 27116.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288c (yeast)
#14: DNA chain DNA (88-MER)


Mass: 27154.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288c (yeast)

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Non-polymers , 4 types, 15 molecules

#15: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#16: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#17: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#18: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1The MCM-ORC (MO) loading intermediateCOMPLEX#1-#140MULTIPLE SOURCES
2MCM-Orc6N lobe of the MCM-ORC (MO) origin licensing intermediate.COMPLEX#6-#141MULTIPLE SOURCESMap derived from multibody refinement in RELION-3.
3Orc1-5-Orc6C lobe of the MCM-ORC (MO) origin licensing intermediateCOMPLEX#1-#6, #13-#141MULTIPLE SOURCESMap derived from multibody refinement in RELION-3.
4The MCM-ORC (MO) loading intermediate protein complexCOMPLEX#1-#121RECOMBINANTRecombinant complex
5DNACOMPLEX#13-#141RECOMBINANTSynthetic DNA
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111.08 MDaNO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
14Saccharomyces cerevisiae S288C (yeast)559292
25Saccharomyces cerevisiae S288C (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
14Saccharomyces cerevisiae S288C (yeast)559292
25synthetic construct (others)32630
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
210 mMMagnesium acetate1
3100 mMPotassium acetate1
41 mMDTT1
50.75 mMATPAdenosine triphosphate1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 288 K
Details: 10 second incubation, 3.5 seconds single side blotting.

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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 FIELDBright-field microscopy / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.68 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
4RELION3CTF correction
5Gctf1.18CTF correction
8UCSF Chimera1.11.2model fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177637 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
15ZR1

5zr1

1
26EYC

6eyc

1
35BK4

5bk4

1
46F0L

6f0l

1

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