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- EMDB-4047: Cryo-EM structure of ternary deletion mutant of human APC/C-Cdh1-... -

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Basic information

Entry
Database: EMDB / ID: EMD-4047
TitleCryo-EM structure of ternary deletion mutant of human APC/C-Cdh1-Hsl1 complex without Apc1 WD40 domain
Map data
Sample
  • Complex: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsLi Q / Chang L / Yang J / Zhang Z / Barford D
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.
Authors: Qiuhong Li / Leifu Chang / Shintaro Aibara / Jing Yang / Ziguo Zhang / David Barford /
Abstract: The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for ...The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.
History
DepositionJul 7, 2016-
Header (metadata) releaseOct 5, 2016-
Map releaseOct 5, 2016-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4047.png

Downloads & links

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Map

FileDownload / File: emd_4047.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.18531242 - 0.3971233
Average (Standard dev.)0.0022356252 (±0.016142555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-131-131-131
Dimensions264264264
Spacing264264264
CellA=B=C: 359.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z359.040359.040359.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-131-131-131
NC/NR/NS264264264
D min/max/mean-0.1850.3970.002

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Supplemental data

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Sample components

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Entire : Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain

EntireName: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Components
  • Complex: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain

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Supramolecule #1: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain

SupramoleculeName: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFU
Molecular weightTheoretical: 1.2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

Concentration0.12 mg/mL
BufferpH: 8
Component:
ConcentrationName
10.0 mMHEPES
150.0 mMNaClSodium chloride
0.5 mMTCEP

Details: Solutions were made fresh from powder and filtered to avoid microbial contamination.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: The grid was coated with continuous carbon film prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 5 seconds before plunging..
DetailsRecombinant protein complex expressed and purified from insect cells. This sample was monodisperse.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 1729 / Average exposure time: 1.1 sec. / Average electron dose: 14.6 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 1.4)
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 71724

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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