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- EMDB-4022: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion... -

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Basic information

Entry
Database: EMDB / ID: EMD-4022
TitleHuman Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
Map dataHuman Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation
Sample
  • Complex: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 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: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4022.png

Downloads & links

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Map

FileDownload / File: emd_4022.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.57 Å/pix.
x 320 pix.
= 502.4 Å		1.57 Å/pix.
x 320 pix.
= 502.4 Å		1.57 Å/pix.
x 320 pix.
= 502.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.571.571.57
CCP4 map header1.571.571.57
EM Navigator Movie #11.271.271.27
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.03517601 - 0.10941491
Average (Standard dev.)0.000709696 (±0.009250927)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 502.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.571.571.57
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z502.400502.400502.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0350.1090.001

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Supplemental data

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Sample components

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Entire : Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion...

EntireName: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
Components
  • Complex: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.

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Supramolecule #1: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion...

SupramoleculeName: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
type: complex / ID: 1 / Parent: 0
Details: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
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

Methodcryo EM
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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsGrafix treated complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 2-17 / Number grids imaged: 1 / Number real images: 5911 / Average exposure time: 1.0 sec. / Average electron dose: 2.35 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 0.0035 µm / Calibrated defocus min: 0.0007 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal magnification: 94000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1142501
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 86398
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)

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