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- EMDB-6334: Negative stain 3D reconstruction of the yeast 26S proteasome in A... -

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Basic information

Entry
Database: EMDB / ID: EMD-6334
TitleNegative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
Map dataNegative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
Sample
  • Sample: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
  • Protein or peptide: 26S proteasomeProteasome
KeywordsProteasome / UPS / Ubp6 / deubiquitinase / regulatory particle
Function / homologyproteasome regulatory particle / Proteasome, subunit alpha/beta
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.2 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 13, 2015-
Map releaseAug 12, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6334.gif

Downloads & links

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Map

FileDownload / File: emd_6334.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 3.9 / Movie #1: 3.9
Minimum - Maximum-26.71676064 - 25.37760544
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-112-112-112
Dimensions224224224
Spacing224224224
CellA=B=C: 918.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z918.400918.400918.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-112-112-112
NC/NR/NS224224224
D min/max/mean-26.71725.378-0.000

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Supplemental data

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Sample components

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Entire : Negative stain 3D reconstruction of the yeast 26S proteasome in A...

EntireName: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
Components
  • Sample: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
  • Protein or peptide: 26S proteasomeProteasome

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Supramolecule #1000: Negative stain 3D reconstruction of the yeast 26S proteasome in A...

SupramoleculeName: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein
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: 1
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Macromolecule #1: 26S proteasome

MacromoleculeName: 26S proteasome / type: protein_or_peptide / ID: 1 / Name.synonym: Proteasome Holoenzyme
Details: Samples of 26S holoenzyme were incubated with wild-type Ubp6 protein in the presence of ATPgS, then diluted to ~25 nM for analysis by negative stain electron microscopy.
Number of copies: 1 / Recombinant expression: No / Database: NCBI
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

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.6
Details: 60 mM HEPES, pH 7.6, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM TCEP, 1 mM ATPgS
StainingType: NEGATIVE
Details: 4 uL 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 ...Details: 4 uL 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

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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.
DateAug 26, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 214 / 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

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Image processing

CTF correctionDetails: Phase flipping of whole micrographs
Final two d classificationNumber classes: 4
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.2 Å / Resolution method: OTHER / Software - Name: Relion
Details: Final 3D models were refined using 9000 particles selected by combining two of the 3D classes from Relion processing.
Number images used: 9000
DetailsThe Appion software package was used for image processing leading to 3D reconstruction. Particles were selected from raw micrographs using the Difference of Gaussians (DoG)-based automated particle picker. The stack was subjected to five rounds of iterative 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). The selected particle stack was subjected to twenty-five iterations of 3D classification, requesting four classes using the Relion suite. Particles comprising well-resolved 3D class averages were used for further refinement by projection matching in Relion.

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