[English] 日本語
Yorodumi
- EMDB-2995: Negative stain 3D reconstruction of the yeast 26S proteasome in c... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-2995
TitleNegative stain 3D reconstruction of the yeast 26S proteasome in complex with ubiquitin-bound Ubp6
Map dataNegative stain 3D reconstruction of yeast 26S holoenzyme in complex with ubiquitin-bound Ubp6
Sample
  • Sample: Yeast 26S proteasome in complex with ubiquitin-bound Ubp6Proteasome
  • Protein or peptide: 26S proteasomeProteasome
  • Protein or peptide: ubiquitin-bound Ubp6
KeywordsProteasome / UPS / Ubp6 / deubiquitinase / regulatory particle
Function / homology
Function and homology information


mitochondria-associated ubiquitin-dependent protein catabolic process / negative regulation of proteasomal protein catabolic process / regulation of proteasomal ubiquitin-dependent protein catabolic process / proteasome regulatory particle / Ub-specific processing proteases / proteasome binding / protein deubiquitination / regulation of proteasomal protein catabolic process / proteasome complex / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity
Similarity search - Function
Ubiquitin carboxyl-terminal hydrolase 14-like / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Proteasome, subunit alpha/beta / Papain-like cysteine peptidase superfamily ...Ubiquitin carboxyl-terminal hydrolase 14-like / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Proteasome, subunit alpha/beta / Papain-like cysteine peptidase superfamily / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin carboxyl-terminal hydrolase 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 22.3 Å
AuthorsBashore C / Dambacher CM / Matyskiela M / Lander GC / Martin A
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome.
Authors: Charlene Bashore / Corey M Dambacher / Ellen A Goodall / Mary E Matyskiela / Gabriel C Lander / Andreas Martin /
Abstract: Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases before substrate processing. In budding yeast, ...Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases before substrate processing. In budding yeast, the deubiquitinase Ubp6 trims ubiquitin chains and affects substrate processing by the proteasome, but the underlying mechanisms and the location of Ubp6 within the holoenzyme have been elusive. Here we show that Ubp6 activity strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Electron microscopy analyses reveal that ubiquitin-bound Ubp6 contacts the N ring and AAA+ ring of the ATPase hexamer and is in proximity to the deubiquitinase Rpn11. Ubiquitin-bound Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as a ubiquitin-dependent 'timer' to coordinate individual processing steps at the proteasome and modulate substrate degradation.
History
DepositionMay 5, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseAug 12, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.77
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.77
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2995.png

Downloads & links

-
Map

FileDownload / File: emd_2995.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D reconstruction of yeast 26S holoenzyme in complex with ubiquitin-bound Ubp6
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 2.77 / Movie #1: 2.77
Minimum - Maximum-20.705484389999999 - 18.307359699999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 787.19995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z787.200787.200787.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-20.70518.3070.000

-
Supplemental data

-
Sample components

-
Entire : Yeast 26S proteasome in complex with ubiquitin-bound Ubp6

EntireName: Yeast 26S proteasome in complex with ubiquitin-bound Ubp6Proteasome
Components
  • Sample: Yeast 26S proteasome in complex with ubiquitin-bound Ubp6Proteasome
  • Protein or peptide: 26S proteasomeProteasome
  • Protein or peptide: ubiquitin-bound Ubp6

-
Supramolecule #1000: Yeast 26S proteasome in complex with ubiquitin-bound Ubp6

SupramoleculeName: Yeast 26S proteasome in complex with ubiquitin-bound Ubp6
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: One to two 19S regulatory particles associates with the core particle to form a functional holoenzyme
Number unique components: 2
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

-
Macromolecule #1: 26S proteasome

MacromoleculeName: 26S proteasome / type: protein_or_peptide / ID: 1 / Name.synonym: Proteasome Holoenzyme
Details: 50uM WT Ubp6 protein was reacted with 75uM ubiquitin vinyl sulfone at 37 degrees. Samples of 26S-bound Ubp6-UbVS were then diluted to ~25nM for analysis by negative stain electron microscopy.
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YYS40 / synonym: Yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa
SequenceGO: proteasome regulatory particle / InterPro: Proteasome, subunit alpha/beta

-
Macromolecule #2: ubiquitin-bound Ubp6

MacromoleculeName: ubiquitin-bound Ubp6 / type: protein_or_peptide / ID: 2
Details: Recombinant Ubp6 was purified from E. coli by Ni affinity chromatography and SEC on a superdex 200. Ubp6-UbVS was made by incubating 75uM UbVS with 50uM Ubp6 at 37 degrees C for 7 hours. ...Details: Recombinant Ubp6 was purified from E. coli by Ni affinity chromatography and SEC on a superdex 200. Ubp6-UbVS was made by incubating 75uM UbVS with 50uM Ubp6 at 37 degrees C for 7 hours. Ubp6-UbVS was added to holoenzymes at a 2:1 ratio and exchanged into 1mM ATPgS.
Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightExperimental: 65 KDa / Theoretical: 65 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceUniProtKB: Ubiquitin carboxyl-terminal hydrolase 6

-
Experimental details

-
Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.6
Details: 60mM HEPES pH 7.6, 50mM NaCl, 50mM KCl, 5mM MgCl2, 0.5mM EDTA, 1mM TCEP, 1mM ATPgS
StainingType: NEGATIVE
Details: 4 microliters of sample was applied to a freshly plasma-cleaned thin carbon surface pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removal of excess protein, negative staining ...Details: 4 microliters of sample was applied to a freshly plasma-cleaned thin carbon surface pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removal of excess protein, negative staining was performed using 2% w/v uranyl formate solution.
GridDetails: 400 mesh Cu-Rh Maxtaform grids were used following deposition of a thin continuous carbon film
VitrificationCryogen name: NONE / Instrument: OTHER

-
Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 52000
Sample stageSpecimen holder: Room temperature, side entry holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 294 K / Max: 297 K / Average: 295 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using a quadrupole stigmator at 52,000 times magnification
DateOct 10, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 357 / Average electron dose: 20 e/Å2
Details: Automated imaging was performed using Leginon software
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

-
Image processing

CTF correctionDetails: Phase flipping of whole micrographs
Final two d classificationNumber classes: 4
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.3 Å / Resolution method: OTHER / Software - Name: Relion
Details: Final 3D models were refined using 12000 particles selected by combining two 3D classes from Relion processing
Number images used: 18565
DetailsAll image processing leading up to 3D reconstruction was performed using the Appion package. Particles were selected using the Difference of Gaussians (DoG)-based automated particle picker from raw micrographs. The stack of particles was subjected to five iterations of 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). Selected 2D classes were used to generate a sub-stack that was subjected to twenty five iterations of 3D classification, requesting four classes using the Relion suite. Particles belonging to well-resolved 3D classes were used for further refinement by projection matching in Relion.

-
Atomic model buiding 1

Initial modelPDB ID:

4cr4

SoftwareName: Chimera
DetailsAn atomic model of yeast Ub-bound Ubp6 was constructed by superimposing the yeast Ubp6 crystal structure PDB 1VJV onto the stucture of the human Rsp14 structure bound to Ubiquitin PDB 2AYO, using the UCSF Chimera MatchMaker tool. These structures have high homology, and the resulting hybrid structure did not exhibit any clashes between the Ubiquitin and Ubp6. This Ubp6-Ub model was docked into the density putatively corresponding to Ubp6. PDB 4CR4 was used for docking other 26S core, base and lid subunits into the map, with the exception of the Rpn8-Rpn11 dimer, for which PDB 4O8Y was used. All docking of PDB structures was performed using the Fit in Map tool of UCSF Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more