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- EMDB-5013: A 3D EM map of the subcomplex Orc1-5 of the yeast origin recognit... -

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Basic information

Entry
Database: EMDB / ID: EMD-5013
TitleA 3D EM map of the subcomplex Orc1-5 of the yeast origin recognition complex (ORC).
Map dataThis is a 3D negatively stained EM map of a subcomplex (Orc1-5) of the yeast origin recognition complex (ORC - Orc1-6). This subcomplex does not contain the smallest subunit Orc6 of ORC.
Sample
  • Sample: Yeast ORC subcomplex Orc1-5.
  • Protein or peptide: Chromosomal replication origin recognition protein Orc1p
  • Protein or peptide: Orc2
  • Protein or peptide: Orc3
  • Protein or peptide: Orc4
  • Protein or peptide: Orc5
KeywordsNegative stain electron microscopy / single particle image reconstruction / yeast replication initiation / origin recognition complex / DNA binding protein
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsChen Z / Speck C / Wendel P / Tang C / Stillman B / Li H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.
Authors: Zhiqiang Chen / Christian Speck / Patricia Wendel / Chunyan Tang / Bruce Stillman / Huilin Li /
Abstract: The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit ...The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC-Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1-5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit-subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1-DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.
History
DepositionMay 8, 2008-
Header (metadata) releaseMay 14, 2008-
Map releaseApr 23, 2009-
UpdateJul 8, 2011-
Current statusJul 8, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5013_1.gif

Downloads & links

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Map

FileDownload / File: emd_5013.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D negatively stained EM map of a subcomplex (Orc1-5) of the yeast origin recognition complex (ORC - Orc1-6). This subcomplex does not contain the smallest subunit Orc6 of ORC.
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour Level1: 2.0 / Movie #1: 2
Minimum - Maximum-0.0000278656 - 9.865919999999999
Average (Standard dev.)0.0892259 (±0.530543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 254 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z254.000254.000254.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS100100100
D min/max/mean-0.0009.8660.089

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Supplemental data

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Sample components

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Entire : Yeast ORC subcomplex Orc1-5.

EntireName: Yeast ORC subcomplex Orc1-5.
Components
  • Sample: Yeast ORC subcomplex Orc1-5.
  • Protein or peptide: Chromosomal replication origin recognition protein Orc1p
  • Protein or peptide: Orc2
  • Protein or peptide: Orc3
  • Protein or peptide: Orc4
  • Protein or peptide: Orc5

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Supramolecule #1000: Yeast ORC subcomplex Orc1-5.

SupramoleculeName: Yeast ORC subcomplex Orc1-5. / type: sample / ID: 1000
Details: The sample was purified by gel filtration and was indeed monodisperse.
Oligomeric state: One heteropentamer (Orc1-Orc2-Orc3-Orc4-Orc5)
Number unique components: 5
Molecular weightTheoretical: 362 KDa

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Macromolecule #1: Chromosomal replication origin recognition protein Orc1p

MacromoleculeName: Chromosomal replication origin recognition protein Orc1p
type: protein_or_peptide / ID: 1 / Name.synonym: Orc1p / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Cell: Yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa
Recombinant expressionOrganism: Sf9 insect cells / Recombinant plasmid: bvORC1

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Macromolecule #2: Orc2

MacromoleculeName: Orc2 / type: protein_or_peptide / ID: 2 / Name.synonym: Orc2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Strain: S. cerevisiae / Cell: Yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 71 KDa / Theoretical: 71 KDa
Recombinant expressionOrganism: Sf9 insect cells / Recombinant plasmid: bvORC2

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Macromolecule #3: Orc3

MacromoleculeName: Orc3 / type: protein_or_peptide / ID: 3 / Name.synonym: Orc3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Strain: S. cerevisiae / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 62 KDa / Theoretical: 62 KDa
Recombinant expressionOrganism: Sf9 insect cells / Recombinant plasmid: bvORC3

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Macromolecule #4: Orc4

MacromoleculeName: Orc4 / type: protein_or_peptide / ID: 4 / Name.synonym: Orc4 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Strain: S. cerevisiae / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa
Recombinant expressionOrganism: bvORC4 / Recombinant plasmid: SF9 insect cells

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Macromolecule #5: Orc5

MacromoleculeName: Orc5 / type: protein_or_peptide / ID: 5 / Name.synonym: Orc5 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Strain: S. cerevisiae / Cell: yeast / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightExperimental: 53 KDa / Theoretical: 53 KDa
Recombinant expressionOrganism: bvORC5 / Recombinant plasmid: SF9 insect cells

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.6
Details: 50 mM Hepes-KOH pH 7.6, 100 mM potassium glutamate, 5 mM MgCl2, 1 mM EGTA, and 1mM ATP-gamma-S
StainingType: NEGATIVE
Details: A 6.0 micro liter drop of sample solution was applied to a glow-discharged 300-mesh copper grid covered with a thin layer of carbon film, and after a brief incubation of 30 - 60 s, the ...Details: A 6.0 micro liter drop of sample solution was applied to a glow-discharged 300-mesh copper grid covered with a thin layer of carbon film, and after a brief incubation of 30 - 60 s, the excess sample solution was blotted with a small piece of filter paper. The grid was then stained in a deep stain procedure by three consecutive 5 micro liter drops of 2.0% uranyl acetate aqueous solution. Each drop was left on grid for 15 - 20 s at room temperature before blotting. After blotting the last stain drop, the grid was quickly dried by a stream of argon to prevent crystallization of the stain salt.
GridDetails: 300 mesh copper grid covered with continuous carbon film
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1200EX
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 5.6 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 250,000 times magnification
Legacy - Electron beam tilt params: 2
DateOct 1, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 50 / Average electron dose: 10 e/Å2 / Od range: 1.3 / Bits/pixel: 14

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 100
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, SPIDER / Number images used: 8935

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