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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: 5013
TitleA 3D EM map of the subcomplex Orc1-5 of the yeast origin recognition complex (ORC).
KeywordsNegative stain electron microscopy / single particle image reconstruction / yeast replication initiation / origin recognition complex / DNA binding protein
SampleYeast ORC subcomplex Orc1-5.
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.
Methodsingle particle reconstruction, at 25 Å resolution
AuthorsChen Z / Speck C / Wendel P / Tang C / Stillman B / Li H
CitationProc. Natl. Acad. Sci. U.S.A., 2008, 105, 10326-10331

Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 10326-10331 Yorodumi Papers
The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.
Zhiqiang Chen / Christian Speck / Patricia Wendel / Chunyan Tang / Bruce Stillman / Huilin Li

DateDeposition: May 8, 2008 / Header (metadata) release: May 14, 2008 / Map release: Apr 23, 2009 / Last update: Jul 8, 2011

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF CHIMERA
  • Download
3D viewer


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Supplemental images

Downloads & links

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Map

Fileemd_5013.map.gz (map file in CCP4 format, 3908 KB)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
100 pix
2.54 Å/pix.
= 254. Å
100 pix
2.54 Å/pix.
= 254. Å
100 pix
2.54 Å/pix.
= 254. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 2.54 Å
Density
Contour Level:2, 2 (movie #1):
Minimum - Maximum-2.78656e-05 - 9.86592
Average (Standard dev.)0.0892259 (0.530543)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions100100100
Origin-50-50-50
Limit494949
Spacing100100100
CellA=B=C: 254 Å
α=β=γ: 90 deg.

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.
Details: The sample was purified by gel filtration and was indeed monodisperse.
Number of components: 5
Oligomeric State: One heteropentamer (Orc1-Orc2-Orc3-Orc4-Orc5)
MassTheoretical: 362 kDa

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Component #1: protein, Chromosomal replication origin recognition protein Orc1p

ProteinName: Chromosomal replication origin recognition protein Orc1p
a.k.a: Orc1p / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 120 kDa / Experimental: 120 kDa
Source (engineered)Expression System: Sf9 insect cells / Vector: bvORC1
Source (natural)Organelle: Nucleus / Location in cell: Nucleus / Cell: Yeast

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Component #2: protein, Orc2

ProteinName: Orc2 / a.k.a: Orc2 / Oligomeric Details: monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 71 kDa / Experimental: 71 kDa
SourceStrain: S. cerevisiae
Source (engineered)Expression System: Sf9 insect cells / Vector: bvORC2
Source (natural)Organelle: Nucleus / Location in cell: Nucleus / Cell: Yeast

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Component #3: protein, Orc3

ProteinName: Orc3 / a.k.a: Orc3 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 62 kDa / Experimental: 62 kDa
SourceStrain: S. cerevisiae
Source (engineered)Expression System: Sf9 insect cells / Vector: bvORC3
Source (natural)Organelle: Nucleus / Location in cell: Nucleus / Cell: yeast

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Component #4: protein, Orc4

ProteinName: Orc4 / a.k.a: Orc4 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 56 kDa / Experimental: 56 kDa
SourceStrain: S. cerevisiae
Source (engineered)Expression System: Bvorc4 / Vector: SF9 insect cells
Source (natural)Organelle: Nucleus / Location in cell: Nucleus / Cell: yeast

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Component #5: protein, Orc5

ProteinName: Orc5 / a.k.a: Orc5 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 53 kDa / Experimental: 53 kDa
SourceStrain: S. cerevisiae
Source (engineered)Expression System: Bvorc5 / Vector: SF9 insect cells
Source (natural)Organelle: Nucleus / Location in cell: Nucleus / Cell: yeast

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.1 mg/ml
Buffer solution: 50 mM Hepes-KOH pH 7.6, 100 mM potassium glutamate, 5 mM MgCl2, 1 mM EGTA, and 1mM ATP-gamma-S
pH: 7.6
Support film300 mesh copper grid covered with continuous carbon film
StainingA 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.
VitrificationInstrument: NONE / Cryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: JEOL 1200EX / Date: Oct 1, 2005
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 10 e/Å2 / Electron beam tilt params: 2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal)
Astigmatism: objective lens astigmatism was corrected at 250,000 times magnification
Cs: 5.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 1500 nm
Specimen HolderHolder: Eucentric / Model: OTHER
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 50 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 12.7 microns / Bit depth: 14 / OD range: 1.3

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 100 / Number of projections: 8935 / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Random Conical Tilt / Software: EMAN, SPIDER / CTF correction: Each micrograph / Resolution: 25 Å / Resolution method: FSC 0.5

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