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- EMDB-6479: Structure of the yeast 26S proteasome lid sub-complex -

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Basic information

Entry
Database: EMDB / ID: EMD-6479
TitleStructure of the yeast 26S proteasome lid sub-complex
Map dataReconstruction of the yeast proteasome lid sub-complex
Sample
  • Sample: Recombinant yeast 26S proteasome lid complex
  • Protein or peptide: 26S proteasome lid sub-complex
KeywordsProteasome / deubiquitinase / Rpn11 / protein homeostasis
Function / homology
Function and homology information


SAGA complex localization to transcription regulatory region / Metalloprotease DUBs / peroxisome fission / proteasome storage granule assembly / transcription export complex 2 / protein deneddylation / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / proteasome regulatory particle ...SAGA complex localization to transcription regulatory region / Metalloprotease DUBs / peroxisome fission / proteasome storage granule assembly / transcription export complex 2 / protein deneddylation / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / proteasome regulatory particle / proteasome regulatory particle, lid subcomplex / mitochondrial fission / metal-dependent deubiquitinase activity / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Regulation of PTEN stability and activity / CDK-mediated phosphorylation and removal of Cdc6 / proteasome binding / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / Neddylation / regulation of protein catabolic process / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / proteasome storage granule / Antigen processing: Ubiquitination & Proteasome degradation / proteasome assembly / protein deubiquitination / Ub-specific processing proteases / mRNA export from nucleus / enzyme regulator activity / protein folding chaperone / Neutrophil degranulation / proteasome complex / double-strand break repair via homologous recombination / metallopeptidase activity / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / molecular adaptor activity / regulation of cell cycle / structural molecule activity / positive regulation of transcription by RNA polymerase II / mitochondrion / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Rpn9, C-terminal helix / Rpn9 C-terminal helix / : / 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 / : ...Rpn9, C-terminal helix / Rpn9 C-terminal helix / : / 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 / PSD13 N-terminal repeats / 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 non-ATPase regulatory subunit 13 / 26S Proteasome non-ATPase regulatory subunit 7/8 / : / : / PSMD12/CSN4, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome regulatory subunit Rpn7, N-terminal / 26S proteasome subunit RPN7 / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / Proteasome/cyclosome repeat / PCI/PINT associated module / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / : / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / TPR repeat region circular profile. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / TPR repeat profile. / MPN domain / MPN domain profile. / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
26S proteasome complex subunit SEM1 / 26S proteasome regulatory subunit RPN12 / 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
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsDambacher CM / Worden EJ / Herzik MA / Martin A / Lander GC
CitationJournal: Elife / Year: 2016
Title: Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition.
Authors: Corey M Dambacher / Evan J Worden / Mark A Herzik / Andreas Martin / Gabriel C Lander /
Abstract: The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a ...The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the 'lid' sub-complex. Prior to the lid's incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation.
History
DepositionOct 11, 2015-
Header (metadata) releaseNov 11, 2015-
Map releaseJan 20, 2016-
UpdateMay 11, 2016-
Current statusMay 11, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0442
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0442
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  • Surface view with fitted model
  • Atomic models: PDB-3jck
  • Surface level: 0.0442
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Structure viewerEM map:
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Supplemental images

400_6479.gif

Downloads & links

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Map

FileDownload / File: emd_6479.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the yeast proteasome lid sub-complex
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 160 pix.
= 209.6 Å		1.31 Å/pix.
x 160 pix.
= 209.6 Å		1.31 Å/pix.
x 160 pix.
= 209.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0642 / Movie #1: 0.0442
Minimum - Maximum-0.13327062 - 0.24653329
Average (Standard dev.)0.00072095 (±0.01381083)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 209.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z209.600209.600209.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1330.2470.001

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Supplemental data

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Supplemental map: emd 6479 additional 1.map

Fileemd_6479_additional_1.map
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Supplemental map: emd 6479 additional 2.map

Fileemd_6479_additional_2.map
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Supplemental map: emd 6479 additional 3.map

Fileemd_6479_additional_3.map
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Supplemental map: emd 6479 half map 1.map

Fileemd_6479_half_map_1.map
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Supplemental map: emd 6479 half map 2.map

Fileemd_6479_half_map_2.map
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Sample components

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Entire : Recombinant yeast 26S proteasome lid complex

EntireName: Recombinant yeast 26S proteasome lid complex
Components
  • Sample: Recombinant yeast 26S proteasome lid complex
  • Protein or peptide: 26S proteasome lid sub-complex

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Supramolecule #1000: Recombinant yeast 26S proteasome lid complex

SupramoleculeName: Recombinant yeast 26S proteasome lid complex / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: 9 subunits / Number unique components: 1
Molecular weightExperimental: 370 KDa / Theoretical: 370 KDa / Method: SDS protein gels and size exclusion chromatography

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Macromolecule #1: 26S proteasome lid sub-complex

MacromoleculeName: 26S proteasome lid sub-complex / type: protein_or_peptide / ID: 1 / Name.synonym: lid
Details: Lid complex was recombinantly expressed in E. coli and purified by size exclusion chromatography.
Number of copies: 1 / Oligomeric state: Heterononamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288C / synonym: Yeast / Location in cell: cytoplasm
Molecular weightExperimental: 370 KDa / Theoretical: 370 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET, pCOLA, pACYC
SequenceGO: proteasome regulatory particle, lid subcomplex / InterPro: Proteasome/cyclosome repeat

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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

Concentration2.5 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES, 100 mM NaCl, 100 mM KCl, 1 mM TCEP
GridDetails: Sample was applied directly to plasma-cleaned holey carbon C-flat grids (400 mesh, 1.2 micrometer holes).
VitrificationCryogen name: ETHANE / Chamber humidity: 88 % / Chamber temperature: 85 K / Instrument: HOMEMADE PLUNGER / Details: Manual plunging was performed in a cold room.
Method: 4 microliters of sample was applied to the grid, blotted for 2 seconds, and plunged into liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 85 K / Max: 90 K / Average: 87.5 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at a nominal magnification of 22,500.
DetailsMicrographs were collected in super-resolution mode with a total frame count of 38 and total exposure time of 7.6 seconds.
DateFeb 10, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 3432 / Average electron dose: 43.8 e/Å2
Details: Micrographs were collected as movies using super-resolution mode with the Gatan K2 Summit direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38168 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImage pre-processing was performed using Appion. 3D classification and reconstruction was performed with RELION.
CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: Appion, CTFFIND3, FindEM, RELION
Details: 3D classification was performed to identify the best 109,396 particles from an initial data set of 254,112.
Number images used: 109396
FSC plot (resolution estimation)

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