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- PDB-8eam: SsoMCM hexamer bound to Mg/ADP-BeFx and DNA. Class 2. Merged part... -

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Basic information

Entry
Database: PDB / ID: 8eam
TitleSsoMCM hexamer bound to Mg/ADP-BeFx and DNA. Class 2. Merged particles from datasets with 3 different DNA entities
Components
  • DNA
  • Minichromosome maintenance protein MCM
KeywordsREPLICATION / TRANSFERASE/DNA / Helicase / ATPase / TRANSFERASE-DNA complex
Function / homology
Function and homology information


MCM complex / DNA duplex unwinding / helicase activity / single-stranded DNA binding / DNA helicase / DNA replication / ATP hydrolysis activity / ATP binding / identical protein binding
Similarity search - Function
: / MCM protein C-terminal winged helix-turn-helix domain / 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 protein C-terminal winged helix-turn-helix domain / 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 / 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
Chem-08T / DNA / DNA (> 10) / Minichromosome maintenance protein MCM
Similarity search - Component
Biological speciesSaccharolobus solfataricus P2 (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsMeagher, M. / Myasnikov, A. / Enemark, E.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136313 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098771 United States
CitationJournal: Int J Mol Sci / Year: 2022
Title: Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation.
Authors: Martin Meagher / Alexander Myasnikov / Eric J Enemark /
Abstract: A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated ...A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.
History
DepositionAug 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Minichromosome maintenance protein MCM
B: Minichromosome maintenance protein MCM
C: Minichromosome maintenance protein MCM
D: Minichromosome maintenance protein MCM
E: Minichromosome maintenance protein MCM
F: Minichromosome maintenance protein MCM
X: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)417,91621
Polymers415,4577
Non-polymers2,45814
Water21612
1


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

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Components

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Protein / DNA chain , 2 types, 7 molecules ABCDEFX

#1: Protein
Minichromosome maintenance protein MCM /


Mass: 68641.961 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus P2 (archaea)
Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: MCM, SSO0774 / Plasmid: pEE078.1 / Production host: Escherichia coli (E. coli) / Variant (production host): -RIPL / References: UniProt: Q9UXG1, DNA helicase
#2: DNA chain DNA /


Mass: 3605.356 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 26 molecules

#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: Zn
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-08T / [[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl]oxy-tris(fluoranyl)beryllium


Mass: 492.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H14BeF3N5O10P2 / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: H2O

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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: SsoMCM hexamer bound to Mg/ADP-BeFx and DNA. Class 2. Merged particles from datasets with 3 different DNA entities.
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Saccharolobus solfataricus P2 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pEE078.1
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 78 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARC2.14.2CTF correction
7PHENIX1.20.1_4487model fitting
9cryoSPARC2.14.2initial Euler assignment
10cryoSPARC3.31final Euler assignmentHomo_Refine_New
11cryoSPARC3.31classificationHetero_Refine
12cryoSPARC3.313D reconstructionHomo_Refine_New
13PHENIX1.20.1_4487model refinementphenix.real_speace_refine
CTF correctionDetails: Initial CTF was calculated by cryoSPARC Patch CTF. CTF parameters were refined during final homogeneous refinement in cryoSPARC.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Particles were merged from 3 different datasets that differ in the specific DNA present. The particles for each individual structure were selected in equivalent ways: Particles were selected ...Details: Particles were merged from 3 different datasets that differ in the specific DNA present. The particles for each individual structure were selected in equivalent ways: Particles were selected from a subset of micrographs with cryoSPARC blob picker. 2D class averages were calculated, selected and used as templates with cryoSPARC template picker.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1493596 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Bond distance and angle restraints for a tetrahedral Zn2+ coordination were applied. Bond distance and angle restraints for a octahedral Mg2+ coordination were applied.
Atomic model buildingPDB-ID: 6MII

6mii


Pdb chain-ID: B
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00224624
ELECTRON MICROSCOPYf_angle_d0.45333352
ELECTRON MICROSCOPYf_dihedral_angle_d6.2863464
ELECTRON MICROSCOPYf_chiral_restr0.0443848
ELECTRON MICROSCOPYf_plane_restr0.0034235

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