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- PDB-7w1y: Human MCM double hexamer bound to natural DNA duplex (polyAT/polyTA) -

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Basic information

Entry
Database: PDB / ID: 7w1y
TitleHuman MCM double hexamer bound to natural DNA duplex (polyAT/polyTA)
Components
  • (DNA (49-MER)) x 2
  • (DNA replication licensing factor ...) x 5
  • Isoform 2 of DNA replication licensing factor MCM3
KeywordsCELL CYCLE/DNA / replication / CELL CYCLE-DNA complex
Function / homology
Function and homology information


Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of phosphorylation / CMG complex / MCM complex / double-strand break repair via break-induced replication ...Switching of origins to a post-replicative state / Unwinding of DNA / nuclear origin of replication recognition complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of phosphorylation / CMG complex / 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 / DNA replication origin binding / cochlea development / Activation of the pre-replicative complex / DNA replication initiation / Activation of ATR in response to replication stress / cellular response to interleukin-4 / DNA helicase activity / cellular response to epidermal growth factor stimulus / Assembly of the pre-replicative complex / helicase activity / Orc1 removal from chromatin / cellular response to xenobiotic stimulus / nucleosome assembly / single-stranded DNA binding / histone binding / DNA helicase / chromosome, telomeric region / DNA replication / cell population proliferation / cilium / intracellular membrane-bounded organelle / apoptotic process / DNA damage response / chromatin / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
DNA replication licensing factor MCM2-like, winged-helix domain / : / 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 MCM2-like, winged-helix domain / : / 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 / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / 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 MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsLi, J. / Dong, J. / Dang, S. / Zhai, Y.
Funding support Hong Kong, 1items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC) Hong Kong
CitationJournal: Cell / Year: 2023
Title: The human pre-replication complex is an open complex.
Authors: Jian Li / Jiangqing Dong / Weitao Wang / Daqi Yu / Xinyu Fan / Yan Chit Hui / Clare S K Lee / Wai Hei Lam / Nathan Alary / Yang Yang / Yingyi Zhang / Qian Zhao / Chun-Long Chen / Bik-Kwoon ...Authors: Jian Li / Jiangqing Dong / Weitao Wang / Daqi Yu / Xinyu Fan / Yan Chit Hui / Clare S K Lee / Wai Hei Lam / Nathan Alary / Yang Yang / Yingyi Zhang / Qian Zhao / Chun-Long Chen / Bik-Kwoon Tye / Shangyu Dang / Yuanliang Zhai /
Abstract: In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this ...In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.
History
DepositionNov 21, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: Isoform 2 of 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
A: DNA replication licensing factor MCM2
B: Isoform 2 of DNA replication licensing factor MCM3
C: DNA replication licensing factor MCM4
D: DNA replication licensing factor MCM5
E: DNA replication licensing factor MCM6
F: DNA replication licensing factor MCM7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,140,05748
Polymers1,133,34514
Non-polymers6,71234
Water00
1


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

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Components

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DNA replication licensing factor ... , 5 types, 10 molecules 2A4C5D6E7F

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


Mass: 102034.102 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P49736
#3: Protein DNA replication licensing factor MCM4 / CDC21 homolog / P1-CDC21


Mass: 96684.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P33991, DNA helicase
#4: Protein DNA replication licensing factor MCM5 / CDC46 homolog / P1-CDC46


Mass: 82406.633 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P33992, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / p105MCM


Mass: 93010.273 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: Q14566, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / CDC47 homolog / P1.1-MCM3


Mass: 81411.875 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P33993, DNA helicase

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Protein , 1 types, 2 molecules 3B

#2: Protein Isoform 2 of DNA replication licensing factor MCM3 / DNA polymerase alpha holoenzyme-associated protein P1 / P1-MCM3 / RLF subunit beta / p102


Mass: 96043.320 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P25205, DNA helicase

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DNA chain , 2 types, 2 molecules

#7: DNA chain DNA (49-MER)


Mass: 15302.170 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Since the register of origin DNA engaged with endogenous hMCM-DHs is heterogenous, it is likely that there is a mix of purines and pyrimidines at each nucleotide position. For this reason we ...Details: Since the register of origin DNA engaged with endogenous hMCM-DHs is heterogenous, it is likely that there is a mix of purines and pyrimidines at each nucleotide position. For this reason we could not build the origin DNA sequence with certainty. Instead, two superposed polyA:polyT and polyT:polyA duplexes, in which all atoms is set at 50 % occupancy, were modelled to surrogate for the heterogenous sequences.
Source: (natural) Homo sapiens (human)
#8: DNA chain DNA (49-MER)


Mass: 14860.490 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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Non-polymers , 4 types, 34 molecules

#9: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Zn
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#11: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#12: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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
1human Mini-chromosome maintenance complexCOMPLEX#1-#80RECOMBINANT
2DNA replication licensing factor MCM 2/3/4/5/6/7COMPLEX#1-#61RECOMBINANT
3DNACOMPLEX#7-#81NATURAL
Molecular weightValue: 1.09 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human) / Cell: HeLa
Buffer solutionpH: 7.5
Details: Solutions were made fresh from concentrated to avoid microbial contamination
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mML-GlutamateL-Glu1
250 mMHEPESHEPES/KOH1
38 mMMagnesium chlorideMgCl21
41 mMEthylene Diamine Tetraacetic AcidEDTA1
50.02 %NP-40NP-401
63 mMAdenosine TriphosphateATP1
72 mMSodium fluorideNaF1
81 mMSodium vanadateNa3VO41
91 mMPhenylmethanesulfonyl fluoridePMSF1
101 *Protein inhibitor cocktailPIC1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.78 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3710
Details: Movies are collected in movie-mode containing 40 frames.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchv0.56particle selection
2EPU2.10.0image acquisition
4Gctfv1.18CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11cryoSPARCv3.0.1initial Euler assignment
12cryoSPARCv3.0.1final Euler assignment
14cryoSPARCv3.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 808142
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329847 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3JA8

3ja8


Accession code: 3JA8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01367948
ELECTRON MICROSCOPYf_angle_d0.97492212
ELECTRON MICROSCOPYf_dihedral_angle_d27.34310059
ELECTRON MICROSCOPYf_chiral_restr0.0610431
ELECTRON MICROSCOPYf_plane_restr0.00511602

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