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- EMDB-5429: Negative stain 3D EM of MCM2-7 from Encephalitozoon cuniculi -

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Basic information

Entry
Database: EMDB / ID: EMD-5429
TitleNegative stain 3D EM of MCM2-7 from Encephalitozoon cuniculi
Map dataSingle-particle 3D reconstruction of EcuMCM2-7 with ATPgammaS
Sample
  • Sample: Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS
  • Protein or peptide: Minichromosome maintenance protein 2
  • Protein or peptide: Minichromosome maintenance protein 3
  • Protein or peptide: Minichromosome maintenance protein 4
  • Protein or peptide: Minichromosome maintenance protein 5
  • Protein or peptide: Minichromosome maintenance protein 6
  • Protein or peptide: Minichromosome maintenance protein 7
KeywordsDNA replication / replicative helicase / MCM2-7 / ATPase / hexameric motor
Biological speciesEncephalitozoon cuniculi (fungus)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsLyubimov AY / Costa A / Bleichert F / Botchan MR / Berger JM
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote.
Authors: Artem Y Lyubimov / Alessandro Costa / Franziska Bleichert / Michael R Botchan / James M Berger /
Abstract: The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is ...The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems.
History
DepositionJun 11, 2012-
Header (metadata) releaseJun 29, 2012-
Map releaseJul 11, 2012-
UpdateJul 11, 2012-
Current statusJul 11, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5429.png

Downloads & links

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Map

FileDownload / File: emd_5429.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle 3D reconstruction of EcuMCM2-7 with ATPgammaS
Voxel sizeX=Y=Z: 3.56 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 1.8
Minimum - Maximum-3.18536615 - 7.23052835
Average (Standard dev.)0.00000743 (±0.99999636)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 227.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.563.563.56
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z227.840227.840227.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-3.1857.2310.000

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Supplemental data

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Sample components

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Entire : Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS

EntireName: Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS
Components
  • Sample: Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS
  • Protein or peptide: Minichromosome maintenance protein 2
  • Protein or peptide: Minichromosome maintenance protein 3
  • Protein or peptide: Minichromosome maintenance protein 4
  • Protein or peptide: Minichromosome maintenance protein 5
  • Protein or peptide: Minichromosome maintenance protein 6
  • Protein or peptide: Minichromosome maintenance protein 7

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Supramolecule #1000: Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS

SupramoleculeName: Encephalitozoon cuniculi MCM2-7 complex bound to ATPgammaS
type: sample / ID: 1000 / Oligomeric state: MCM2-7 heterohexamer / Number unique components: 6
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: Minichromosome maintenance protein 2

MacromoleculeName: Minichromosome maintenance protein 2 / type: protein_or_peptide / ID: 1 / Name.synonym: Mcm2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 88 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #2: Minichromosome maintenance protein 3

MacromoleculeName: Minichromosome maintenance protein 3 / type: protein_or_peptide / ID: 2 / Name.synonym: Mcm3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 77 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #3: Minichromosome maintenance protein 4

MacromoleculeName: Minichromosome maintenance protein 4 / type: protein_or_peptide / ID: 3 / Name.synonym: Mcm4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 80 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #4: Minichromosome maintenance protein 5

MacromoleculeName: Minichromosome maintenance protein 5 / type: protein_or_peptide / ID: 4 / Name.synonym: Mcm5 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 79 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #5: Minichromosome maintenance protein 6

MacromoleculeName: Minichromosome maintenance protein 6 / type: protein_or_peptide / ID: 5 / Name.synonym: Mcm6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 82 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #6: Minichromosome maintenance protein 7

MacromoleculeName: Minichromosome maintenance protein 7 / type: protein_or_peptide / ID: 6 / Name.synonym: Mcm7 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Molecular weightTheoretical: 77 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 8
Details: 300 mM NaOAc, 50 mM imidazole, pH 8.0, 5 mM Mg(OAc)2, 0.2 mM TCEP, 10% glycerol, 10 mM ATPgammaS
StainingType: NEGATIVE
Details: Grids with adsorbed protein (diluted to 0.03 mg/mL) floated on 2% uranyl formate for 10 sec (x 5 times)
GridDetails: 400 mesh copper grids, nitrocellulose amyl acetate support, glow discharged, holey
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 5, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Number real images: 16532 / Average electron dose: 20 e/Å2

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX
Details: Nine iterative rounds were performed, with the output of each round used as starting model (after filtering) for the subsequent round until convergence. Convergence was determined by lack of ...Details: Nine iterative rounds were performed, with the output of each round used as starting model (after filtering) for the subsequent round until convergence. Convergence was determined by lack of further changes in volume features and distortion of FSC at higher resolution.
Number images used: 16532
DetailsParticles selected automatically using DoG Picker and Batchboxer in the APPION environment

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Atomic model buiding 1

Initial modelPDB ID:

3f9v

SoftwareName: Chimera
DetailsProtocol: Rigid body. The 6 protomers were fit using sequential volume-to-volume fitting in Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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