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- PDB-8s0b: H. sapiens MCM bound to double stranded DNA and ORC6 as part of t... -

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Basic information

Entry
Database: PDB / ID: 8s0b
TitleH. sapiens MCM bound to double stranded DNA and ORC6 as part of the MCM-ORC complex
Components
  • (DNA (45-mer)) x 2
  • (DNA replication licensing factor ...) x 6
  • Origin recognition complex subunit 6
KeywordsREPLICATION / AAA+ ATPase / DNA helicase
Function / homology
Function and homology information


CDC6 association with the ORC:origin complex / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / mitotic DNA replication / CMG complex / regulation of phosphorylation ...CDC6 association with the ORC:origin complex / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / mitotic DNA replication / CMG complex / regulation of phosphorylation / MCM complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / cochlea development / DNA unwinding involved in DNA replication / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / cellular response to interleukin-4 / Activation of ATR in response to replication stress / DNA helicase activity / Assembly of the ORC complex at the origin of replication / helicase activity / Assembly of the pre-replicative complex / fibrillar center / Orc1 removal from chromatin / cellular response to xenobiotic stimulus / nucleosome assembly / single-stranded DNA binding / histone binding / DNA helicase / DNA replication / cell population proliferation / chromosome, telomeric region / centrosome / DNA damage response / chromatin / apoptotic process / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, metazoa/plant / : / ORC6 second cyclin-like domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / 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 ...Origin recognition complex, subunit 6, metazoa/plant / : / ORC6 second cyclin-like domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / 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 / 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 / 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 / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM6 / Origin recognition complex subunit 6
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsGreiwe, J.F. / Weissmann, F. / Diffley, J.F.X. / Costa, A.
Funding support United Kingdom, European Union, 4items
OrganizationGrant numberCountry
The Francis Crick InstituteCC2002, CC2009 United Kingdom
European Research Council (ERC)820102European Union
Wellcome Trust219527/Z/19/Z United Kingdom
European Research Council (ERC)101020432-MeChroRepEuropean Union
Citation
Journal: Nature / Year: 2024
Title: MCM Double Hexamer Loading Visualised with Human Proteins
Authors: Weissmann, F. / Greiwe, J.F. / Puehringer, T. / Eastwood, E.L. / Couves, E.C. / Miller, T.C.R. / Diffley, J.F.X. / Costa, A.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionFeb 13, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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: DNA replication licensing factor MCM5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
X: DNA (45-mer)
Y: DNA (45-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)605,92126
Polymers602,5969
Non-polymers3,32417
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules F

#1: Protein Origin recognition complex subunit 6


Mass: 28017.697 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC6, ORC6L / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y5N6

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DNA replication licensing factor ... , 6 types, 6 molecules 234567

#2: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2 homolog / Nuclear protein BM28


Mass: 102034.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2, BM28, CCNL1, CDCL1, KIAA0030 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P49736, DNA helicase
#3: Protein DNA replication licensing factor MCM3 / DNA polymerase alpha holoenzyme-associated protein P1 / P1-MCM3 / RLF subunit beta / p102


Mass: 91297.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P25205, DNA helicase
#4: Protein DNA replication licensing factor MCM4 / CDC21 homolog / P1-CDC21


Mass: 96702.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4, CDC21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P33991, DNA helicase
#5: Protein DNA replication licensing factor MCM5 / CDC46 homolog / P1-CDC46


Mass: 82406.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5, CDC46 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P33992, DNA helicase
#6: Protein DNA replication licensing factor MCM6 / p105MCM


Mass: 93010.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14566, DNA helicase
#7: Protein DNA replication licensing factor MCM7 / CDC47 homolog / P1.1-MCM3


Mass: 81411.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P33993, DNA helicase

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DNA chain , 2 types, 2 molecules XY

#8: DNA chain DNA (45-mer)


Mass: 13837.883 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: DNA chain DNA (45-mer)


Mass: 13877.905 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 17 molecules

#10: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, 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

Component
IDNameTypeEntity IDParent-IDSource
1H. sapiens MCM bound to double stranded DNA and ORC6 as part of the MCM-ORC complexCOMPLEX#1-#90MULTIPLE SOURCES
2H. sapiens ORC6COMPLEX#11RECOMBINANT
3Double stranded DNACOMPLEX#8-#91RECOMBINANT
4H. sapiens MCM2-7COMPLEX#2-#71RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33synthetic construct (others)32630
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
44Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The MCM recruitment reaction was reconstituted in vitro using purified H. sapiens proteins, a short DNA template and ATPgammaS. Four microlitres of the entire reaction was applied on a grid ...Details: The MCM recruitment reaction was reconstituted in vitro using purified H. sapiens proteins, a short DNA template and ATPgammaS. Four microlitres of the entire reaction was applied on a grid and incubated for 1 min at room temperature before blotting with filter paper for 5 s and plunge-freezing in liquid ethane.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K

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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 / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 9.4 sec. / Electron dose: 49.28 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 31569
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182341 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDDetailsSource nameTypeAccession codeChain-IDInitial refinement model-ID
1Model of the DNA-bound MCM hexamer that was determined in the same dataset.Otherexperimental model
2AlphaFoldin silico modelAF-Q9Y5N6-F1F2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 91.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002733138
ELECTRON MICROSCOPYf_angle_d0.490745133
ELECTRON MICROSCOPYf_chiral_restr0.03985144
ELECTRON MICROSCOPYf_plane_restr0.00385476
ELECTRON MICROSCOPYf_dihedral_angle_d16.36815274

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