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- EMDB-1526: Single particle reconstruction of Methanothermobacter thermautotr... -

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Basic information

Entry
Database: EMDB / ID: EMD-1526
TitleSingle particle reconstruction of Methanothermobacter thermautotrophicus MCM protein bound to a 5,600bp dsDNA fragment.
Map dataThis is an electron density map of the MthMCM helicase treated with a 5,600 bp dsDNA fragment.
Sample
  • Sample: Methanothermobacter thermautotrophicus MCM protein bound to a 5,600 bp dsDNA fragment
  • DNA: DNA
  • Protein or peptide: Mini-chromosome Maintenance complexMinichromosome maintenance
Keywordshelicase / AAA+ ATPase / DNA replication
Biological speciessynthetic construct (others) / Methanothermobacter thermautotrophicus str. Delta H (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsCosta A / van Duinen G / Medagli B / Chong J / Sakakibara N / Kelman Z / Nair SK / Patwardhan A / Onesti S
CitationJournal: EMBO J / Year: 2008
Title: Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase.
Authors: Alessandro Costa / Gijs van Duinen / Barbara Medagli / James Chong / Nozomi Sakakibara / Zvi Kelman / Satish K Nair / Ardan Patwardhan / Silvia Onesti /
Abstract: The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize ...The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.
History
DepositionJun 20, 2008-
Header (metadata) releaseJun 23, 2008-
Map releaseApr 2, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1526.gif

emd_1526.png

emd_1526.tif

Downloads & links

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Map

FileDownload / File: emd_1526.map.gz / Format: CCP4 / Size: 3.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an electron density map of the MthMCM helicase treated with a 5,600 bp dsDNA fragment.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.6 Å/pix.
x 95 pix.
= 332.8 Å		2.6 Å/pix.
x 95 pix.
= 332.8 Å		2.6 Å/pix.
x 95 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.6 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.135 / Movie #1: 0.11
Minimum - Maximum-0.721348 - 0.569189
Average (Standard dev.)0.00198268 (±0.0751419)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-47-46-47
Dimensions959595
Spacing959595
CellA=B=C: 332.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S123
start NC/NR/NS-46-47-47
NC/NR/NS959595
D min/max/mean-0.7210.5690.002

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Supplemental data

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Sample components

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Entire : Methanothermobacter thermautotrophicus MCM protein bound to a 5,6...

EntireName: Methanothermobacter thermautotrophicus MCM protein bound to a 5,600 bp dsDNA fragment
Components
  • Sample: Methanothermobacter thermautotrophicus MCM protein bound to a 5,600 bp dsDNA fragment
  • DNA: DNA
  • Protein or peptide: Mini-chromosome Maintenance complexMinichromosome maintenance

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Supramolecule #1000: Methanothermobacter thermautotrophicus MCM protein bound to a 5,6...

SupramoleculeName: Methanothermobacter thermautotrophicus MCM protein bound to a 5,600 bp dsDNA fragment
type: sample / ID: 1000
Oligomeric state: One homohexamer of MCM binds to one dsDNA fragment
Number unique components: 2

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Macromolecule #1: DNA

MacromoleculeName: DNA / type: dna / ID: 1 / Name.synonym: 5,600 bp pET15b vector fragment / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: synthetic construct (others)

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Macromolecule #2: Mini-chromosome Maintenance complex

MacromoleculeName: Mini-chromosome Maintenance complex / type: protein_or_peptide / ID: 2 / Name.synonym: MCM / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Methanothermobacter thermautotrophicus str. Delta H (archaea)
Strain: Delta H
Molecular weightTheoretical: 450 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.012 mg/mL
BufferpH: 7.5
Details: 30mM Tris HCl pH 7.5, 50 mM NaCl, 5mM MgCl2, 5 mM beta-mercaptoethanol
GridDetails: Quantifoil R 1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4.5 K / Instrument: OTHER / Details: Vitrification instrument: FEI Vitrobot plunger
Method: Blot for 2.5 seconds at an offset of -1 before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN
Image recordingDigitization - Scanner: OTHER / Digitization - Sampling interval: 6.5 µm / Number real images: 22 / Average electron dose: 17 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 800
Final angle assignmentDetails: beta 0 degrees, gamma 90 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: IMAGIC-5 / Number images used: 10010
DetailsParticles were selected automatically using Boxer on defocal pair images

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