EMDB-3034: Structure of the 26S proteasome-Ubp6 complex

Basic information

• Entry: Database: EMDB / ID: EMD-3034
• Title: Structure of the 26S proteasome-Ubp6 complex
• Map data: Reconstruction of the 26S proteasome in presence of Ubp6 and ubiquitin aldehyde

Sample

• Sample: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehydeProteasome
• Protein or peptide: 26S ProteasomeProteasome
• Protein or peptide: Ubp6
• Protein or peptide: ubiqutin aldehyde

• Keywords: conformational switching / protein degradation / proteostasis / quality control / Ubp6

Function / homology

Function:

SAGA complex localization to transcription regulatory region / mitochondria-associated ubiquitin-dependent protein catabolic process / peroxisome fission / negative regulation of proteasomal protein catabolic process / regulation of proteasomal ubiquitin-dependent protein catabolic process / proteasome storage granule assembly / transcription export complex 2 / proteasome regulatory particle assembly / protein deneddylation / nonfunctional rRNA decay / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / proteasome regulatory particle / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / protein-containing complex localization / mitochondrial fission / proteasome regulatory particle, base subcomplex / K48-linked polyubiquitin modification-dependent protein binding / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ub-specific processing proteases / peptide catabolic process / proteasome binding / regulation of protein catabolic process / protein deubiquitination / Peptide chain elongation / Selenocysteine synthesis / proteasome storage granule / Formation of a pool of free 40S subunits / Eukaryotic Translation Termination / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / Viral mRNA Translation / polyubiquitin modification-dependent protein binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / endopeptidase activator activity / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / Major pathway of rRNA processing in the nucleolus and cytosol / proteasome assembly / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / regulation of proteasomal protein catabolic process / enzyme regulator activity / mRNA export from nucleus / : / protein folding chaperone / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / Endosomal Sorting Complex Required For Transport (ESCRT) / NOTCH2 Activation and Transmission of Signal to the Nucleus / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / Regulation of FZD by ubiquitination / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Neutrophil degranulation / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / proteasome complex

Domain/homology:

Ubiquitin carboxyl-terminal hydrolase 14-like / Proteasome subunit Rpn10 / Rpn9, C-terminal helix / Rpn9 C-terminal helix / Proteasomal ubiquitin receptor Rpn13/ADRM1 / Proteasomal ubiquitin receptor Rpn13/ADRM1, Pru domain superfamily / Rpn13/ADRM1, Pru domain / Proteasome complex subunit Rpn13, Pru domain / Pru (pleckstrin-like receptor for ubiquitin) domain profile. / : / 26S proteasome regulatory subunit RPN7/PSMD6 C-terminal helix / 26S proteasome non-ATPase regulatory subunit Rpn12 / 26S proteasome regulatory subunit, C-terminal / Proteasome regulatory subunit C-terminal / DSS1/SEM1 / 26S proteasome regulatory subunit RPN5, C-terminal domain / : / DSS1/SEM1 family / 26S proteasome regulatory subunit RPN5 C-terminal domain / 26S proteasome subunit RPN2, N-terminal domain / DSS1_SEM1 / 26S proteasome regulatory subunit Rpn6, N-terminal / 6S proteasome subunit Rpn6, C-terminal helix domain / 26S proteasome regulatory subunit RPN6 N-terminal domain / 26S proteasome subunit RPN6 C-terminal helix domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn2/Psmd1 subunit / 26S Proteasome non-ATPase regulatory subunit 13 / 26S proteasome regulatory subunit RPN2, C-terminal / 26S proteasome regulatory subunit RPN2 C-terminal domain / 26S Proteasome non-ATPase regulatory subunit 7/8 / : / 26S proteasome regulatory subunit 7, OB domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn1 subunit / RPN1, N-terminal / 26S proteasome non-ATPase regulatory subunit RPN1, C-terminal / RPN1 N-terminal domain / 26S proteasome non-ATPase regulatory subunit RPN1 C-terminal / 26S proteasome regulatory subunit Rpn7, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome subunit RPN7 / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / Proteasome/cyclosome repeat / Proteasome/cyclosome repeat / PCI/PINT associated module / von Willebrand factor type A domain / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / HEAT repeats / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile. / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Ribosomal L40e family / VWFA domain profile. / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Proteasome subunit / Proteasome, subunit alpha/beta / von Willebrand factor, type A / AAA ATPase, AAA+ lid domain / AAA+ lid domain / TPR repeat region circular profile. / ATPase, AAA-type, conserved site / AAA-protein family signature. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / TPR repeat profile.

Component:

26S proteasome regulatory subunit RPN13 / 26S proteasome complex subunit SEM1 / Polyubiquitin-C / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-6 / Proteasome subunit beta type-2 / Proteasome subunit beta type-3 / Proteasome subunit beta type-5 / Proteasome subunit beta type-7 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit RPN12 / 26S proteasome regulatory subunit RPN2 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit beta type-1 / 26S proteasome regulatory subunit RPN1 / 26S proteasome regulatory subunit RPN10 / 26S proteasome regulatory subunit RPN3 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / Ubiquitin carboxyl-terminal hydrolase RPN11 / Ubiquitin carboxyl-terminal hydrolase 6 / 26S proteasome subunit RPT4 / Ubiquitin-ribosomal protein eL40 fusion protein / 26S proteasome regulatory subunit 8 homolog / 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 species: Saccharomyces cerevisiae (brewer's yeast) / Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 9.5 Å
• Authors: Aufderheide A / Beck F / Stengel F / Hartwig M / Schweitzer A / Pfeifer G / Goldberg AL / Sakata E / Baumeister W / Foerster F
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2015; Title: Structural characterization of the interaction of Ubp6 with the 26S proteasome.; Authors: Antje Aufderheide / Florian Beck / Florian Stengel / Michaela Hartwig / Andreas Schweitzer / Günter Pfeifer / Alfred L Goldberg / Eri Sakata / Wolfgang Baumeister / Friedrich Förster / ; Abstract: In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.

History

Deposition	Jun 1, 2015	-
Header (metadata) release	Jul 8, 2015	-
Map release	Jul 15, 2015	-
Update	Aug 12, 2015	-
Current status	Aug 12, 2015	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3034.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstruction of the 26S proteasome in presence of Ubp6 and ubiquitin aldehyde
• Voxel size: X=Y=Z: 1.99 Å

Density

Contour Level	By AUTHOR: 1.17 / Movie #1: 1.17
Minimum - Maximum	-11.709941860000001 - 12.581256870000001
Average (Standard dev.)	0.00614737 (±0.297102)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 280 280 280 Spacing 280 280 280
Cell	A=B=C: 557.2 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.99	1.99	1.99
M x/y/z	280	280	280
origin x/y/z	0.000	0.000	0.000
length x/y/z	557.200	557.200	557.200
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-147	-147	-146
NX/NY/NZ	294	294	294
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	280	280	280
D min/max/mean	-11.710	12.581	0.006

Supplemental data

Sample components

Entire : 26S Proteasome from Saccharomyces cerevisiae in the presence of S...

• Entire: Name: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehydeProteasome

Components

• Sample: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehydeProteasome
• Protein or peptide: 26S ProteasomeProteasome
• Protein or peptide: Ubp6
• Protein or peptide: ubiqutin aldehyde

Supramolecule #1000: 26S Proteasome from Saccharomyces cerevisiae in the presence of S...

• Supramolecule: Name: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde; type: sample / ID: 1000 / Number unique components: 3
• Molecular weight: Experimental: 2.6 MDa / Theoretical: 2.6 MDa

Macromolecule #1: 26S Proteasome

• Macromolecule: Name: 26S Proteasome / type: protein_or_peptide / ID: 1 / Recombinant expression: No
• Source (natural): Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
• Molecular weight: Experimental: 2.6 MDa / Theoretical: 2.6 MDa

Macromolecule #2: Ubp6

• Macromolecule: Name: Ubp6 / type: protein_or_peptide / ID: 2 / Oligomeric state: Monomer / Recombinant expression: Yes
• Source (natural): Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
• Molecular weight: Theoretical: 60 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)
• Sequence: UniProtKB: Ubiquitin carboxyl-terminal hydrolase 6

Macromolecule #3: ubiqutin aldehyde

• Macromolecule: Name: ubiqutin aldehyde / type: protein_or_peptide / ID: 3 ; Details: ubiquitin aldehyde was purchased from BostonBiochem (Cat.#U-211); Recombinant expression: No
• Source (natural): Organism: Homo sapiens (human) / synonym: human

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.35 mg/mL
• Vitrification: Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Alignment procedure: Legacy - Electron beam tilt params: 0
• Date: Dec 12, 2014
• Image recording: Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 5630 / Average electron dose: 45 e/Ų / Details: Every image is the average of 7 aligned frames.
• Tilt angle min: 0
• Tilt angle max: 0
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Details: micrograph
• Final two d classification: Number classes: 29100
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: OTHER / Software - Name: xmipp / Number images used: 53000
• Details: The particles were selected using an automatic selection program. Each physical 26S particles was considered as two particles for processing according to pseudo-C2 symmetry.