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- EMDB-2873: Negative stain electron microscopy asymmetric reconstruction of h... -

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Basic information

Entry
Database: EMDB / ID: EMD-2873
TitleNegative stain electron microscopy asymmetric reconstruction of human minichromosome maintenance bound to a DNA substrate
Map dataReconstruction of hMCM bound DNA
Sample
  • Sample: Recombinant human mini chromosome maintenance complex bound to a DNA substrate
  • Protein or peptide: human minichromosome maintenance (2-7) complex
Keywordsminichromosome maintenance / MCM2-7 / hMCM / DNA helicase / eukaryote
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: 1.679108764
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.679108764
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

4.tif

Downloads & links

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Map

FileDownload / File: emd_2873.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of hMCM bound DNA
Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 1.6791088
Minimum - Maximum-0.75365782 - 5.00809717
Average (Standard dev.)0.02831108 (±0.2496849)
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-0.7545.0080.028

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Supplemental data

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Sample components

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Entire : Recombinant human mini chromosome maintenance complex bound to a ...

EntireName: Recombinant human mini chromosome maintenance complex bound to a DNA substrate
Components
  • Sample: Recombinant human mini chromosome maintenance complex bound to a DNA substrate
  • Protein or peptide: human minichromosome maintenance (2-7) complex

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Supramolecule #1000: Recombinant human mini chromosome maintenance complex bound to a ...

SupramoleculeName: Recombinant human mini chromosome maintenance complex bound to a DNA substrate
type: sample / ID: 1000 / Oligomeric state: heterohexamer / Number unique components: 1
Molecular weightTheoretical: 567 KDa

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Macromolecule #1: human minichromosome maintenance (2-7) complex

MacromoleculeName: human minichromosome maintenance (2-7) complex / type: protein_or_peptide / ID: 1 / Name.synonym: hMCM, MCM2-7 / 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, 200 mM sodium glutamate, 1 mM DTT, 0.1 mM AEBSF
StainingType: NEGATIVE / Details: Stained with methylamine tungstate, pH 7
GridDetails: Carbon coated grid
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: BRIGHT FIELDBright-field microscopy
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: 72

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Image processing

CTF correctionDetails: EACH MICROGRAPH
Final two d classificationNumber classes: 155
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: IMAGIC / Number images used: 1550
DetailsParticles were selected using Boxer

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