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- EMDB-3136: A Single Alpha Helix Drives Extensive Remodeling of the Proteasom... -

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Basic information

Entry
Database: EMDB / ID: EMD-3136
TitleA Single Alpha Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly
Map data
SampleRecombinant LP2 (Lid Particle 2)
  • (26S proteasome regulatory subunit ...) x 8
Keywordsproteasome / ubiquitin / proteolysis / electron microscopy / mass spectrometry
Function / homology
Function and homology information


protein deneddylation => GO:0000338 / molecular_function / SAGA complex localization to transcription regulatory region / proteasome storage granule assembly / peroxisome fission / filamentous growth / transcription export complex 2 / maintenance of DNA trinucleotide repeats / protein deneddylation / histone deubiquitination ...protein deneddylation => GO:0000338 / molecular_function / SAGA complex localization to transcription regulatory region / proteasome storage granule assembly / peroxisome fission / filamentous growth / transcription export complex 2 / maintenance of DNA trinucleotide repeats / protein deneddylation / histone deubiquitination / mitochondrial fission / COP9 signalosome / nuclear proteasome complex / proteasome regulatory particle / proteasome regulatory particle, lid subcomplex / Lys63-specific deubiquitinase activity / proteasome binding / regulation of protein catabolic process / proteasome storage granule / proteasome complex / thiol-dependent ubiquitin-specific protease activity / proteasome assembly / mRNA export from nucleus / enzyme regulator activity / metallopeptidase activity / ubiquitinyl hydrolase 1 / double-strand break repair via homologous recombination / regulation of cell cycle / proteasome-mediated ubiquitin-dependent protein catabolic process / ubiquitin-dependent protein catabolic process / protein deubiquitination / structural molecule activity / mitochondrion / metal ion binding / nucleus / cytosol / cytoplasm
26S proteasome non-ATPase regulatory subunit 3 / Winged helix-like DNA-binding domain superfamily / 26S Proteasome non-ATPase regulatory subunit 7/8 / Rpn11/EIF3F, C-terminal / Tetratricopeptide repeat / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome regulatory subunit, C-terminal / Tetratricopeptide repeat-containing domain / Tetratricopeptide-like helical domain superfamily / DSS1/SEM1 ...26S proteasome non-ATPase regulatory subunit 3 / Winged helix-like DNA-binding domain superfamily / 26S Proteasome non-ATPase regulatory subunit 7/8 / Rpn11/EIF3F, C-terminal / Tetratricopeptide repeat / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome regulatory subunit, C-terminal / Tetratricopeptide repeat-containing domain / Tetratricopeptide-like helical domain superfamily / DSS1/SEM1 / Proteasome component (PCI) domain / JAB1/MPN/MOV34 metalloenzyme domain / 26S Proteasome non-ATPase regulatory subunit 12 / 26S Proteasome non-ATPase regulatory subunit 13 / 26S Proteasome non-ATPase regulatory subunit 6 / 26S Proteasome non-ATPase regulatory subunit 14 / 6S proteasome subunit Rpn6, C-terminal helix domain / MPN domain / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / 26S proteasome regulatory subunit Rpn6, N-terminal / Rpn9, C-terminal helix / 26S proteasome regulatory subunit RPN5, C-terminal domain / Winged helix DNA-binding domain superfamily / in:IPR013143: / ArsR-like helix-turn-helix domain / 26S proteasome regulatory subunit, C-terminal / DSS1/SEM1 / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / Rpn11/EIF3F, C-terminal / Proteasome component (PCI) domain / JAB1/MPN/MOV34 metalloenzyme domain / Tetratricopeptide repeat-containing domain
26S proteasome complex subunit SEM1 / 26S proteasome regulatory subunit RPN3 / Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome regulatory subunit RPN9 / 26S proteasome regulatory subunit RPN7 / 26S proteasome regulatory subunit RPN8 / 26S proteasome regulatory subunit RPN5 / 26S proteasome regulatory subunit RPN6
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 16 Å
AuthorsTomko Jr RJ / Taylor DW / Chen ZA / Wang HW / Rappsilber J / Hochstrasser M
CitationJournal: Cell / Year: 2015
Title: A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly.
Authors: Robert J Tomko / David W Taylor / Zhuo A Chen / Hong-Wei Wang / Juri Rappsilber / Mark Hochstrasser /
Abstract: Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis ...Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes.
History
DepositionAug 31, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseOct 21, 2015-
UpdateOct 21, 2015-
Current statusOct 21, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3136.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 160 pix.
= 448. Å
2.8 Å/pix.
x 160 pix.
= 448. Å
2.8 Å/pix.
x 160 pix.
= 448. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-19.906469349999998 - 23.58402061
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 448.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z448.000448.000448.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-19.90623.584-0.000

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Supplemental data

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Sample components

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Entire Recombinant LP2 (Lid Particle 2)

EntireName: Recombinant LP2 (Lid Particle 2) / Details: The sample was monodisperse. / Number of components: 8
Oligomeric State: 1 Rpn3: 1 Rpn5: 1 Rpn6: 1 Rpn7: 1 Rpn8: 1 Rpn9: 1 Rpn11: 1 Sem1
MassTheoretical: 343 kDa

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Component #1: protein, 26S proteasome regulatory subunit RPN3

ProteinName: 26S proteasome regulatory subunit RPN3 / a.k.a: RPN3 / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 64 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesInterPro: 26S proteasome regulatory subunit, C-terminal, Proteasome component (PCI) domain
UniProt: 26S proteasome regulatory subunit RPN3
Gene Ontology: ubiquitin-dependent protein catabolic process, regulation of protein catabolic process, molecular_function, proteasome complex, proteasome regulatory particle, lid subcomplex

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Component #2: protein, 26S proteasome regulatory subunit RPN5

ProteinName: 26S proteasome regulatory subunit RPN5 / a.k.a: RPN5 / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 52 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesGene Ontology: ubiquitin-dependent protein catabolic process, protein deneddylation => GO:0000338, molecular_function, proteasome complex, proteasome regulatory particle, lid subcomplex
InterPro: Proteasome component (PCI) domain, ArsR-like helix-turn-helix domain
UniProt: 26S proteasome regulatory subunit RPN5

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Component #3: protein, 26S proteasome regulatory subunit RPN6

ProteinName: 26S proteasome regulatory subunit RPN6 / a.k.a: RPN6 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 50 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesUniProt: 26S proteasome regulatory subunit RPN6
InterPro: Proteasome component (PCI) domain, InterPro: IPR013143
Gene Ontology: ubiquitin-dependent protein catabolic process, structural molecule activity, proteasome complex, proteasome regulatory particle, lid subcomplex

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Component #4: protein, 26S proteasome regulatory subunit RPN7

ProteinName: 26S proteasome regulatory subunit RPN7 / a.k.a: RPN7 / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesUniProt: 26S proteasome regulatory subunit RPN7
InterPro: 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1, Proteasome component (PCI) domain, Tetratricopeptide repeat-containing domain
Gene Ontology: ubiquitin-dependent protein catabolic process, structural molecule activity, proteasome complex, proteasome regulatory particle, lid subcomplex

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Component #5: protein, 26S proteasome regulatory subunit RPN8

ProteinName: 26S proteasome regulatory subunit RPN8 / a.k.a: RPN8 / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 38 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesGene Ontology: ubiquitin-dependent protein catabolic process, molecular_function, proteasome complex, proteasome regulatory particle, lid subcomplex
UniProt: 26S proteasome regulatory subunit RPN8
InterPro: JAB1/MPN/MOV34 metalloenzyme domain, Rpn11/EIF3F, C-terminal

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Component #6: protein, 26S proteasome regulatory subunit RPN9

ProteinName: 26S proteasome regulatory subunit RPN9 / a.k.a: RPN9 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 46 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesUniProt: 26S proteasome regulatory subunit RPN9
Gene Ontology: ubiquitin-dependent protein catabolic process, structural molecule activity, proteasome complex, proteasome regulatory particle, lid subcomplex
InterPro: Proteasome component (PCI) domain, ArsR-like helix-turn-helix domain

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Component #7: protein, 26S proteasome regulatory subunit RPN11

ProteinName: 26S proteasome regulatory subunit RPN11 / a.k.a: RPN11 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 34 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesInterPro: JAB1/MPN/MOV34 metalloenzyme domain, Rpn11/EIF3F, C-terminal
Gene Ontology: protein deubiquitination, mitochondrial fission, thiol-dependent ubiquitin-specific protease activity, proteasome complex, proteasome regulatory particle, lid subcomplex
UniProt: Ubiquitin carboxyl-terminal hydrolase RPN11

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Component #8: protein, 26S proteasome regulatory subunit Sem1

ProteinName: 26S proteasome regulatory subunit Sem1 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 10 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
External referencesInterPro: DSS1/SEM1 / UniProt: 26S proteasome complex subunit SEM1
Gene Ontology: proteasome complex, proteasome regulatory particle, lid subcomplex, proteasome-mediated ubiquitin-dependent protein catabolic process, proteasome assembly

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.04 mg/mL
Buffer solution: 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, and 10% glycerol
pH: 7.5
Support film400 mesh Cu grid with thin carbon, glow discharged with a sputter coater.
StainingSample was negatively stained with six consecutive droplets of 2% uranyl acetate.
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 20 / Date: Apr 26, 2013 / Details: Data acquired using Leginon.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - 1400 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 600

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 60000
3D reconstructionAlgorithm: back-projection / Software: EMAN2, SPARX / CTF correction: Each micrograph / Resolution: 16 Å / Resolution method: FSC 0.5, semi-independent

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