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- EMDB-2226: Single particle analysis of recombinant human anaphase promoting ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2226
TitleSingle particle analysis of recombinant human anaphase promoting complex (APC/C)
Map datareconstruction of recombinant human APC/C
Sample
  • Sample: recombinant human APC/C
  • Protein or peptide: x 14 types
KeywordsAnaphase promoting complex / cyclosome / cell cycle / recombinant expression / single particle / 3-dimensional structure / ubiquitination
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsZhang Z / Yang J / Kong EH / Chao WCH / Morris EP / da Fonseca PCA / Barford D
CitationJournal: Biochem J / Year: 2013
Title: Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C).
Authors: Ziguo Zhang / Jing Yang / Eric H Kong / William C H Chao / Edward P Morris / Paula C A da Fonseca / David Barford /
Abstract: Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the ...Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.
History
DepositionOct 23, 2012-
Header (metadata) releaseOct 31, 2012-
Map releaseOct 31, 2012-
UpdateDec 26, 2012-
Current statusDec 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.25
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2226.tif

Downloads & links

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Map

FileDownload / File: emd_2226.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of recombinant human APC/C
Voxel sizeX=Y=Z: 3.4725 Å
Density
Contour LevelBy AUTHOR: 3.25 / Movie #1: 3.25
Minimum - Maximum-17.873140339999999 - 19.05537605
Average (Standard dev.)0.05397222 (±0.88403845)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-70-70-70
Dimensions140140140
Spacing140140140
CellA=B=C: 486.15002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.47253.47253.4725
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z486.150486.150486.150
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-70-70-70
NC/NR/NS140140140
D min/max/mean-17.87319.0550.054

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Supplemental data

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Sample components

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Entire : recombinant human APC/C

EntireName: recombinant human APC/C
Components
  • Sample: recombinant human APC/C
  • Protein or peptide: Apc1
  • Protein or peptide: Apc5
  • Protein or peptide: Apc15
  • Protein or peptide: Apc10
  • Protein or peptide: Apc8
  • Protein or peptide: Apc13
  • Protein or peptide: Apc2
  • Protein or peptide: Apc4
  • Protein or peptide: Apc11
  • Protein or peptide: Apc3
  • Protein or peptide: Apc16
  • Protein or peptide: Cdc26
  • Protein or peptide: Apc7
  • Protein or peptide: Apc6

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Supramolecule #1000: recombinant human APC/C

SupramoleculeName: recombinant human APC/C / type: sample / ID: 1000 / Number unique components: 14
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Apc1

MacromoleculeName: Apc1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #2: Apc5

MacromoleculeName: Apc5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #3: Apc15

MacromoleculeName: Apc15 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #4: Apc10

MacromoleculeName: Apc10 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #5: Apc8

MacromoleculeName: Apc8 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #6: Apc13

MacromoleculeName: Apc13 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #7: Apc2

MacromoleculeName: Apc2 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #8: Apc4

MacromoleculeName: Apc4 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #9: Apc11

MacromoleculeName: Apc11 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #10: Apc3

MacromoleculeName: Apc3 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #11: Apc16

MacromoleculeName: Apc16 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #12: Cdc26

MacromoleculeName: Cdc26 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #13: Apc7

MacromoleculeName: Apc7 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #14: Apc6

MacromoleculeName: Apc6 / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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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

BufferpH: 8 / Details: 20 mM HEPES NaOH pH 8.0, 200 mM NaCl, 2 mM DTT
StainingType: NEGATIVE / Details: grids stained with 2% (w/v) uranyl acetate
GridDetails: Quantifoil R1.2/1.3 grids coated with a thin layer of carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 1, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 100 e/Å2
Details: on recording each adjacent boxes of 2x2 pixels were averaged
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: Spider, Imagic / Number images used: 5639
DetailsParticles were manually selected using the EMAN boxer software. The images were analysed using a combination of IMAGIC and Spider software.

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