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- EMDB-2929: Mechanism of Ubiquitin Ligation to a Substrate by human Anaphase-... -

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Basic information

Entry
Database: EMDB / ID: EMD-2929
TitleMechanism of Ubiquitin Ligation to a Substrate by human Anaphase-Promoting Complex
Map dataanaphase promoting complex
Sample
  • Sample: Cdh1 and UbcH10 bound to Anaphase Promoting Complex
  • Protein or peptide: Anaphase Promoting Complex
KeywordsAnaphase Promoting Complex / ubiquitin ligation
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsBrown N / VanderLinden R / Watson E / Qiao R / Grace CR / Yamaguchi M / Weissmann F / Frye J / Dube P / Cho SE ...Brown N / VanderLinden R / Watson E / Qiao R / Grace CR / Yamaguchi M / Weissmann F / Frye J / Dube P / Cho SE / Actis M / Rodrigues P / Fujii N / Peters JM / Schulman BA / Stark H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.
Authors: Nicholas G Brown / Ryan VanderLinden / Edmond R Watson / Renping Qiao / Christy R R Grace / Masaya Yamaguchi / Florian Weissmann / Jeremiah J Frye / Prakash Dube / Shein Ei Cho / Marcelo L ...Authors: Nicholas G Brown / Ryan VanderLinden / Edmond R Watson / Renping Qiao / Christy R R Grace / Masaya Yamaguchi / Florian Weissmann / Jeremiah J Frye / Prakash Dube / Shein Ei Cho / Marcelo L Actis / Patrick Rodrigues / Naoaki Fujii / Jan-Michael Peters / Holger Stark / Brenda A Schulman /
Abstract: For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered ...For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2∼Ub catalytic modules such as APC11-UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10∼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
History
DepositionMar 10, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseApr 15, 2015-
UpdateMay 13, 2015-
Current statusMay 13, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 50
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 50
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2929.png

Downloads & links

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Map

FileDownload / File: emd_2929.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationanaphase promoting complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.57 Å/pix.
x 256 pix.
= 401.92 Å		1.57 Å/pix.
x 256 pix.
= 401.92 Å		1.57 Å/pix.
x 256 pix.
= 401.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.57 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 50.0 / Movie #1: 50
Minimum - Maximum-77.057800290000003 - 177.376434329999995
Average (Standard dev.)0.00011701 (±10.151549340000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 401.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.571.571.57
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z401.920401.920401.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-77.058177.3760.000

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Supplemental data

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Sample components

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Entire : Cdh1 and UbcH10 bound to Anaphase Promoting Complex

EntireName: Cdh1 and UbcH10 bound to Anaphase Promoting Complex
Components
  • Sample: Cdh1 and UbcH10 bound to Anaphase Promoting Complex
  • Protein or peptide: Anaphase Promoting Complex

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Supramolecule #1000: Cdh1 and UbcH10 bound to Anaphase Promoting Complex

SupramoleculeName: Cdh1 and UbcH10 bound to Anaphase Promoting Complex / type: sample / ID: 1000 / Number unique components: 3
Molecular weightExperimental: 1.5 MDa

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Macromolecule #1: Anaphase Promoting Complex

MacromoleculeName: Anaphase Promoting Complex / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human

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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 / Details: 50 mM Hepes, 150 mM NaCl, 2 mM MgCl2
GridDetails: 200 mesh copper grids, quantifoil with thin carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateFeb 18, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 2097 / Average electron dose: 30 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 94000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 0.0001 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: OTHER / Number images used: 47791

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