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Yorodumi- PDB-6ef2: Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state) -
+Open data
-Basic information
Entry | Database: PDB / ID: 6ef2 | |||||||||
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Title | Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state) | |||||||||
Components |
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Keywords | MOTOR PROTEIN / 26S Proteasome / ATPase / AAA+ / Protease / Ubiquitin | |||||||||
Function / homology | Function and homology information proteasome regulatory particle assembly / nonfunctional rRNA decay / mitotic cell cycle phase transition / cytosolic proteasome complex / proteasome-activating activity / protein-containing complex localization / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network ...proteasome regulatory particle assembly / nonfunctional rRNA decay / mitotic cell cycle phase transition / cytosolic proteasome complex / proteasome-activating activity / protein-containing complex localization / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / 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 storage granule / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome core complex, alpha-subunit complex / : / Neutrophil degranulation / proteasome complex / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / positive regulation of protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / chromatin remodeling / cell division / protein domain specific binding / mRNA binding / ubiquitin protein ligase binding / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.27 Å | |||||||||
Authors | de la Pena, A.H. / Goodall, E.A. / Gates, S.N. / Lander, G.C. / Martin, A. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Science / Year: 2018 Title: Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation. Authors: Andres H de la Peña / Ellen A Goodall / Stephanie N Gates / Gabriel C Lander / Andreas Martin / Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the ...The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26 proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6ef2.cif.gz | 566.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ef2.ent.gz | 448.4 KB | Display | PDB format |
PDBx/mmJSON format | 6ef2.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ef/6ef2 ftp://data.pdbj.org/pub/pdb/validation_reports/ef/6ef2 | HTTPS FTP |
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-Related structure data
Related structure data | 9044MC 9042C 9043C 9045C 6ef0C 6ef1C 6ef3C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Proteasome subunit alpha type- ... , 6 types, 6 molecules ABCDEF
#1: Protein | Mass: 26759.469 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: SCL1, PRC2, PRS2, YGL011C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P21243, proteasome endopeptidase complex |
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#2: Protein | Mass: 27078.670 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PRE8, PRS4, YML092C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P23639, proteasome endopeptidase complex |
#3: Protein | Mass: 26805.178 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PRE9, PRS5, YGR135W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P23638, proteasome endopeptidase complex |
#4: Protein | Mass: 26862.277 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PRE6, YOL038W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P40303, proteasome endopeptidase complex |
#5: Protein | Mass: 27242.596 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PUP2, DOA5, YGR253C, G9155 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P32379, proteasome endopeptidase complex |
#6: Protein | Mass: 25355.631 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PRE5, YMR314W, YM9924.06 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P40302, proteasome endopeptidase complex |
-Protein , 2 types, 2 molecules GL
#7: Protein | Mass: 27092.719 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: PRE10, PRC1, PRS1, YOR362C, O6650 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 References: UniProt: P21242, proteasome endopeptidase complex |
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#12: Protein | Mass: 29353.756 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT4, CRL13, PCS1, SUG2, YOR259C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P53549 |
-26S proteasome regulatory subunit ... , 5 types, 5 molecules HIJKM
#8: Protein | Mass: 30452.154 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT1, CIM5, YTA3, YKL145W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33299 |
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#9: Protein | Mass: 28889.051 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT2, YHS4, YTA5, YDL007W, D2920 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P40327 |
#10: Protein | Mass: 29010.736 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT6, CIM3, CRL3, SUG1, TBPY, TBY1, YGL048C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: Q01939 |
#11: Protein | Mass: 29000.164 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT3, YNT1, YTA2, YDR394W, D9509.14 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33298 |
#13: Protein | Mass: 29845.061 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) Strain: ATCC 204508 / S288c / Gene: RPT5, YTA1, YOR117W, O3258, YOR3258W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33297 |
-Protein/peptide , 1 types, 1 molecules s
#14: Protein/peptide | Mass: 1572.701 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: P07818*PLUS |
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-Non-polymers , 2 types, 6 molecules
#15: Chemical | ChemComp-ATP / #16: Chemical | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 26S proteasomes were diluted to a concentration of 20 micromolar in a buffer with an ATP regeneration system, and 6 mM ortho-phenanthroline. This solution was mixed with an equal volume of ...Details: 26S proteasomes were diluted to a concentration of 20 micromolar in a buffer with an ATP regeneration system, and 6 mM ortho-phenanthroline. This solution was mixed with an equal volume of 50 micromolar ubiquitinated model substrate | ||||||||||||||||||||||||||||||||||||
Specimen support | Details: unspecified | ||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K Details: specimens were manually blotted with Whatman #1 filter paper |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: images were acquired in nanoprobe mode |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1000 nm / Calibrated defocus min: -1500 nm / Calibrated defocus max: -3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 6.25 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11656 |
Image scans | Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 25 / Used frames/image: 1-25 |
-Processing
EM software |
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Image processing | Details: Camera was operated in counting mode | ||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTF correction was performed by Relion during reconstruction Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 579361 Details: Particles were selected using the Relion template-based particle picker | ||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47349 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5MPC Accession code: 5MPC / Source name: PDB / Type: experimental model |