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- EMDB-4027: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Check... -

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Basic information

Entry
Database: EMDB / ID: EMD-4027
TitleHuman Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
Map dataHuman Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
Sample
  • Complex: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsStark H / Schulman BA / Peters JM
CitationJournal: Mol Cell / Year: 2016
Title: Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.
Authors: Masaya Yamaguchi / Ryan VanderLinden / Florian Weissmann / Renping Qiao / Prakash Dube / Nicholas G Brown / David Haselbach / Wei Zhang / Sachdev S Sidhu / Jan-Michael Peters / Holger Stark ...Authors: Masaya Yamaguchi / Ryan VanderLinden / Florian Weissmann / Renping Qiao / Prakash Dube / Nicholas G Brown / David Haselbach / Wei Zhang / Sachdev S Sidhu / Jan-Michael Peters / Holger Stark / Brenda A Schulman /
Abstract: The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). ...The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
History
DepositionJun 12, 2016-
Header (metadata) releaseAug 17, 2016-
Map releaseAug 17, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4027.png

Downloads & links

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Map

FileDownload / File: emd_4027.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.32 Å/pix.
x 160 pix.
= 371.2 Å		2.32 Å/pix.
x 160 pix.
= 371.2 Å		2.32 Å/pix.
x 160 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 25. / Movie #1: 25
Minimum - Maximum-49.808064000000002 - 110.887780000000006
Average (Standard dev.)0.000016183436 (±10.041802000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 371.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.322.322.32
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z371.200371.200371.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-49.808110.8880.000

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Supplemental data

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Sample components

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Entire : Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Check...

EntireName: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
Components
  • Complex: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20

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Supramolecule #1: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Check...

SupramoleculeName: Human Anaphase-Promoting Complex/Cyclosome bound to Mitotic Checkpoint Complex (APC/C-MCC) and the E2 UBE2C (aka UBCH10) poised for ubiquitination of MCC's CDC20
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: biGBac
Molecular weightTheoretical: 1.5 MDa

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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

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHepes
200.0 mMsodium chlorideNaCl
2.0 mMMagnesium chlorideMgCl2
StainingType: NEGATIVE / Material: Uranyl Formate
DetailsGrafix treated complex

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingFilm or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 120560
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Particle selectionNumber selected: 101226
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 21865
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)

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