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- EMDB-1245: Structural analysis of the anaphase-promoting complex reveals mul... -

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Basic information

Entry
Database: EMDB / ID: EMD-1245
TitleStructural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation.
Map dataStructure of Saccharomyces cerevisiae Anaphase-Promoting Complex/Cyclosome (APC). Due to heterogeneity, we obtained 2 structures (A and B) from our dataset. This is "structure B" while EMD-1174 is "structure A".
Sample
  • Sample: Anaphase-Promoting Complex
  • Protein or peptide: Anaphase-Promoting Complex, Cyclosome
Function / homologyanaphase-promoting complex
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsPassmore LA / Booth CR / Venien-Bryan C / Ludtke SJ / Fioretto C / Johnson LN / Chiu W / Barford D
CitationJournal: Mol Cell / Year: 2005
Title: Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation.
Authors: Lori A Passmore / Christopher R Booth / Catherine Vénien-Bryan / Steven J Ludtke / Céline Fioretto / Louise N Johnson / Wah Chiu / David Barford /
Abstract: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the ...The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the cell cycle. Despite its central role in these processes, information concerning its composition and structure is limited. Here, we determined the structure of yeast APC/C by cryo-electron microscopy (cryo-EM). Docking of tetratricopeptide repeat (TPR)-containing subunits indicates that they likely form a scaffold-like outer shell, mediating assembly of the complex and providing potential binding sites for regulators and substrates. Quantitative determination of subunit stoichiometry indicates multiple copies of specific subunits, consistent with a total APC/C mass of approximately 1.7 MDa. Moreover, yeast APC/C forms both monomeric and dimeric species. Dimeric APC/C is a more active E3 ligase than the monomer, with greatly enhanced processivity. Our data suggest that multimerisation and/or the presence of multiple active sites facilitates the APC/C's ability to elongate polyubiquitin chains.
History
DepositionJul 17, 2006-
Header (metadata) releaseJul 18, 2006-
Map releaseJul 18, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.889875985
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.889875985
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1245.gif

Downloads & links

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Map

FileDownload / File: emd_1245.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Saccharomyces cerevisiae Anaphase-Promoting Complex/Cyclosome (APC). Due to heterogeneity, we obtained 2 structures (A and B) from our dataset. This is "structure B" while EMD-1174 is "structure A".
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.17 Å/pix.
x 144 pix.
= 312.192 Å		2.17 Å/pix.
x 144 pix.
= 312.192 Å		2.17 Å/pix.
x 144 pix.
= 312.192 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.168 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 2.45 / Movie #1: 1.889876
Minimum - Maximum-0.718464 - 7.37117
Average (Standard dev.)-0.00000000240875 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 312.192 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1682.1682.168
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z312.192312.192312.192
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-0.7187.371-0.000

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Supplemental data

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Sample components

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Entire : Anaphase-Promoting Complex

EntireName: Anaphase-Promoting Complex
Components
  • Sample: Anaphase-Promoting Complex
  • Protein or peptide: Anaphase-Promoting Complex, Cyclosome

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Supramolecule #1000: Anaphase-Promoting Complex

SupramoleculeName: Anaphase-Promoting Complex / type: sample / ID: 1000
Details: The APC was freshly purified before grid preparation.
Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 1.7 MDa / Theoretical: 1.7 MDa

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Macromolecule #1: Anaphase-Promoting Complex, Cyclosome

MacromoleculeName: Anaphase-Promoting Complex, Cyclosome / type: protein_or_peptide / ID: 1 / Name.synonym: APC
Details: Endogenous APC was purified using a TAP tag present at the C-terminus of the Cdc16 subunit. The APC has 13 different protein subunits. There are multiple copies of specific APC subunits, ...Details: Endogenous APC was purified using a TAP tag present at the C-terminus of the Cdc16 subunit. The APC has 13 different protein subunits. There are multiple copies of specific APC subunits, consistent with a total mass of 1.7 MDa.
Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: CDC16-TAP BJ2168 / synonym: budding yeast
Molecular weightExperimental: 1.7 MDa / Theoretical: 1.7 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceGO: anaphase-promoting complex

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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

Concentration0.125 mg/mL
BufferpH: 8
Details: 20 mM HEPES, 150 mM NaCl, 2 mM EGTA, 3 mM DTT, 1 mM Magnesium acetate, 0.015% (w/v) n-Dodecyl B-D-maltoside (DDM)
GridDetails: Quantifoil R2/2, 200 mesh with an additional layer of freshly floated carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: 4 ul sample was applied to the grid then blotted two times one second on both sides before plunging into liquid ethane.

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid-nitrogen cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 92 K
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 650 / Average electron dose: 15 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Complete phase flipping and amplitude weighting with wiener filtration using the 1D structure factor
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: eman
Details: Due to sample heterogeneity, the data could be separated into two main groups using a multirefinement procedure. Structure A (7500 particles) is in EMD-1174. This is structure B (6800 particles).
Number images used: 19384

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Atomic model buiding 1

SoftwareName: Foldhunter, DockEM
DetailsProtocol: rigid body. 15 or 18 consecutive TPR motifs modelled from PP5 (1A17) were docked as rigid structures into the APC density map using Foldhunter or DockEM. Both programs produced similar results.
RefinementProtocol: RIGID BODY FIT / Target criteria: Cross-correlation

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