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Yorodumi- EMDB-4047: Cryo-EM structure of ternary deletion mutant of human APC/C-Cdh1-... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4047 | |||||||||
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Title | Cryo-EM structure of ternary deletion mutant of human APC/C-Cdh1-Hsl1 complex without Apc1 WD40 domain | |||||||||
Map data | ||||||||||
Sample |
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Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.0 Å | |||||||||
Authors | Li Q / Chang L / Yang J / Zhang Z / Barford D | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4047.map.gz | 7 MB | EMDB map data format | |
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Header (meta data) | emd-4047-v30.xml emd-4047.xml | 12.4 KB 12.4 KB | Display Display | EMDB header |
Images | emd_4047.png | 75 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4047 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4047 | HTTPS FTP |
-Validation report
Summary document | emd_4047_validation.pdf.gz | 215.9 KB | Display | EMDB validaton report |
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Full document | emd_4047_full_validation.pdf.gz | 215.1 KB | Display | |
Data in XML | emd_4047_validation.xml.gz | 6.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4047 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4047 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_4047.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.36 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Entire | Name: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain |
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Components |
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-Supramolecule #1: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Supramolecule | Name: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFU |
Molecular weight | Theoretical: 1.2 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | cell |
-Sample preparation
Concentration | 0.12 mg/mL | ||||||||
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Buffer | pH: 8 Component:
Details: Solutions were made fresh from powder and filtered to avoid microbial contamination. | ||||||||
Grid | Model: 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. | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 5 seconds before plunging.. | ||||||||
Details | Recombinant protein complex expressed and purified from insect cells. This sample was monodisperse. |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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