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- EMDB-10072: cryo EM map of human APC/CCDH1 bound to the avid UbVW_dim trap -

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Basic information

Entry
Database: EMDB / ID: EMD-10072
Titlecryo EM map of human APC/CCDH1 bound to the avid UbVW_dim trap
Map datacryo EM map of human APC/CCDH1 bound to the avid UbVW_dim trap
Sample
  • Complex: Anaphase-Promoting Complex/Cyclosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsWatson ER / Prabu JR / Schulman BA
Funding support Germany, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
European Union227764 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site.
Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / ...Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / Holger Stark / Jan-Michael Peters / Nicholas G Brown / Sachdev S Sidhu / Brenda A Schulman /
Abstract: Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential ...Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.
History
DepositionJun 14, 2019-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 7, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10072.png

Downloads & links

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Map

FileDownload / File: emd_10072.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo EM map of human APC/CCDH1 bound to the avid UbVW_dim trap
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.019727716 - 0.054310914
Average (Standard dev.)0.00032872648 (±0.0021449549)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0200.0540.000

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Supplemental data

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Additional map: None

Fileemd_10072_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: None

Fileemd_10072_additional_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Anaphase-Promoting Complex/Cyclosome

EntireName: Anaphase-Promoting Complex/Cyclosome
Components
  • Complex: Anaphase-Promoting Complex/Cyclosome

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Supramolecule #1: Anaphase-Promoting Complex/Cyclosome

SupramoleculeName: Anaphase-Promoting Complex/Cyclosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 166914

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