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- EMDB-2479: Electron microscopy structure of the Drosophila origin recognitio... -

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Basic information

Entry
Database: EMDB / ID: EMD-2479
TitleElectron microscopy structure of the Drosophila origin recognition complex
Map dataReconstruction of Drosophila ORC in presence of 1mM ATPgS
Sample
  • Sample: Drosophila origin recognition complex with ATPgS
  • Protein or peptide: Orc6
  • Protein or peptide: Orc5
  • Protein or peptide: Orc4
  • Protein or peptide: Orc3
  • Protein or peptide: Orc2
  • Protein or peptide: Orc1
Keywordsorigin recognition complex / DNA replication initiation
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsBleichert F / Balasov M / Chesnokov I / Nogales E / Botchan MR / Berger JM
CitationJournal: Elife / Year: 2013
Title: A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation.
Authors: Franziska Bleichert / Maxim Balasov / Igor Chesnokov / Eva Nogales / Michael R Botchan / James M Berger /
Abstract: In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain ...In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.
History
DepositionSep 26, 2013-
Header (metadata) releaseOct 9, 2013-
Map releaseOct 30, 2013-
UpdateOct 30, 2013-
Current statusOct 30, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2479.tif

Downloads & links

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Map

FileDownload / File: emd_2479.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Drosophila ORC in presence of 1mM ATPgS
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.36 Å/pix.
x 80 pix.
= 348.8 Å		4.36 Å/pix.
x 80 pix.
= 348.8 Å		4.36 Å/pix.
x 80 pix.
= 348.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-9.22742558 - 18.94441986
Average (Standard dev.)0.00002795 (±0.9999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-9.22718.9440.000

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Supplemental data

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Sample components

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Entire : Drosophila origin recognition complex with ATPgS

EntireName: Drosophila origin recognition complex with ATPgS
Components
  • Sample: Drosophila origin recognition complex with ATPgS
  • Protein or peptide: Orc6
  • Protein or peptide: Orc5
  • Protein or peptide: Orc4
  • Protein or peptide: Orc3
  • Protein or peptide: Orc2
  • Protein or peptide: Orc1

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Supramolecule #1000: Drosophila origin recognition complex with ATPgS

SupramoleculeName: Drosophila origin recognition complex with ATPgS / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: heterohexamer / Number unique components: 6
Molecular weightTheoretical: 390 KDa

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Macromolecule #1: Orc6

MacromoleculeName: Orc6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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Macromolecule #2: Orc5

MacromoleculeName: Orc5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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Macromolecule #3: Orc4

MacromoleculeName: Orc4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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Macromolecule #4: Orc3

MacromoleculeName: Orc3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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Macromolecule #5: Orc2

MacromoleculeName: Orc2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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Macromolecule #6: Orc1

MacromoleculeName: Orc1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High5

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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

Concentration0.0116 mg/mL
BufferpH: 7.8
Details: 50mM Tris-HCL pH 7.8, 300mM KCl, 5mM MgCl2, 1mM ATPgS
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 4 drops of 2% uranyl formate for 10 seconds each
GridDetails: 400 mesh copper grid with continuous carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureAverage: 297 K
DateOct 11, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 49000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 30

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Image processing

DetailsParticles were selected using the automated particle selection software DoG Picker. 3D reconstructions were performed with SPIDER.
CTF correctionDetails: each micrograph for untilted, each particle for tilted
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Particles were automatically selected with DoG Picker. The contrast transfer function was estimated with CTFFIND/CTFTILT and phases flipped with SPIDER. Projection-matching refinement was ...Details: Particles were automatically selected with DoG Picker. The contrast transfer function was estimated with CTFFIND/CTFTILT and phases flipped with SPIDER. Projection-matching refinement was performed with SPIDER using a previously determined 3D reconstruction of Drosophila ORC as a starting model.
Number images used: 70000

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