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- EMDB-2872: Negative stain electron microscopy of recombinant human minichrom... -

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Basic information

Entry
Database: EMDB / ID: EMD-2872
TitleNegative stain electron microscopy of recombinant human minichromosome maintenance complex
Map dataReconstruction of recombinant human minichromosome maintenance complex
Sample
  • Sample: Recombinant human mini chromosome maintenance complex 2-7
  • Protein or peptide: Mini chromosome maintenance (2-7) complex
Keywordsmini chromosome maintenance / DNA helicase / MCM2-7 / hMCM
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsHesketh EL / Parker-Manuel RP / Chaban Y / Satti R / Coverley D / Orlova EV / Chong JPJ
CitationJournal: J Biol Chem / Year: 2015
Title: DNA induces conformational changes in a recombinant human minichromosome maintenance complex.
Authors: Emma L Hesketh / Richard P Parker-Manuel / Yuriy Chaban / Rabab Satti / Dawn Coverley / Elena V Orlova / James P J Chong /
Abstract: ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in ...ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.
History
DepositionJan 29, 2015-
Header (metadata) releaseFeb 18, 2015-
Map releaseFeb 18, 2015-
UpdateApr 15, 2015-
Current statusApr 15, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

3.tif

Downloads & links

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Map

FileDownload / File: emd_2872.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of recombinant human minichromosome maintenance complex
Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 1.2
Minimum - Maximum-0.01498062 - 3.9045434
Average (Standard dev.)0.02013654 (±0.18367308)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-92-92-92
Dimensions200200200
Spacing200200200
CellA=B=C: 500.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z500.000500.000500.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-92-92-92
NC/NR/NS200200200
D min/max/mean-0.0153.9050.020

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Supplemental data

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Sample components

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Entire : Recombinant human mini chromosome maintenance complex 2-7

EntireName: Recombinant human mini chromosome maintenance complex 2-7
Components
  • Sample: Recombinant human mini chromosome maintenance complex 2-7
  • Protein or peptide: Mini chromosome maintenance (2-7) complex

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Supramolecule #1000: Recombinant human mini chromosome maintenance complex 2-7

SupramoleculeName: Recombinant human mini chromosome maintenance complex 2-7
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 567 KDa

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Macromolecule #1: Mini chromosome maintenance (2-7) complex

MacromoleculeName: Mini chromosome maintenance (2-7) complex / type: protein_or_peptide / ID: 1 / Name.synonym: MCM, hMCM / Number of copies: 1 / Oligomeric state: heterohexamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 567 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pET-32, PCDF-2, pRSF-2

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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

Concentration0.999 mg/mL
BufferpH: 8
Details: 25 mM HEPES pH 8.0, 200mM sodium glutamate, 1mM DTT, 0.1 mM AEBSF
StainingType: NEGATIVE / Details: Stained with methylamine tungstate
GridDetails: Carbon coated grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 67000 / Illumination mode: SPOT SCAN / Imaging mode: DARK FIELD
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateMay 10, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 31

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Image processing

CTF correctionDetails: EACH MICROGRAPH
Final two d classificationNumber classes: 100
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: IMAGIC / Details: Final maps were averaged from 10 classes / Number images used: 1100
DetailsParticle picking was carried out automatically using the program Boxer EMAN suite (Tang et al., 2007). Analysis of the CTF and correction was completed using the program CTFIT (EMAN suite, Tang et al., 2007). The following image analysis was performed using IMAGIC-5 (van Heel et al., 1996).

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