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- PDB-6ef3: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state) -

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Entry
Database: PDB / ID: 6ef3
TitleYeast 26S proteasome bound to ubiquitinated substrate (4D motor state)
Components
  • (26S proteasome regulatory subunit ...) x 5
  • (Proteasome subunit alpha type- ...) x 6
  • (Proteasome subunit beta type- ...) x 8
  • 26S proteasome subunit RPT4
  • Model substrate polypeptide
  • Probable proteasome subunit alpha type-7
  • Ubiquitin carboxyl-terminal hydrolase RPN11
  • Ubiquitin-60S ribosomal protein L40
KeywordsMOTOR PROTEIN / 26S Proteasome / ATPase / AAA+ / Protease / Motor protein / Ubiquitin
Function / homologyProteasomal ATPase OB C-terminal domain / Proteasome beta-type subunits signature. / Proteasome subunit A N-terminal signature / Proteasome beta subunits C terminal / Maintenance of mitochondrial structure and function / Ubiquitin domain signature. / Proteasome alpha-type subunits signature. / AAA-protein family signature. / Ubiquitin domain profile. / Ribosomal L40e family ...Proteasomal ATPase OB C-terminal domain / Proteasome beta-type subunits signature. / Proteasome subunit A N-terminal signature / Proteasome beta subunits C terminal / Maintenance of mitochondrial structure and function / Ubiquitin domain signature. / Proteasome alpha-type subunits signature. / AAA-protein family signature. / Ubiquitin domain profile. / Ribosomal L40e family / MPN domain profile. / Proteasome alpha-type subunit profile. / Proteasome beta-type subunit profile. / Translesion synthesis by REV1 / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / Cross-presentation of soluble exogenous antigens (endosomes) / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Ubiquitin family / SCF-beta-TrCP mediated degradation of Emi1 / 26S Proteasome regulatory subunit 7 / Proteasome subunit alpha2 / Proteasome subunit alpha4 / Proteasome subunit alpha 1 / Proteasome subunit beta 1 / Proteasome subunit beta 2 / Proteasome subunit beta 7 / 26S Proteasome regulatory subunit 4 / 26S Proteasome regulatory subunit 6B / Proteasome subunit / 26S proteasome regulatory subunit 8 / MPN domain / Proteasome subunit alpha 3 / Proteasome subunit beta Pre3 / Proteasome subunit alpha 7-like / Ribosomal protein L40e superfamily / ATPase family associated with various cellular activities (AAA) / L13a-mediated translational silencing of Ceruloplasmin expression / SRP-dependent cotranslational protein targeting to membrane / Proteasome subunit alpha5 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / rt:r-sce-69229: / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Formation of a pool of free 40S subunits / GTP hydrolysis and joining of the 60S ribosomal subunit / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Gap-filling DNA repair synthesis and ligation in TC-NER / E3 ubiquitin ligases ubiquitinate target proteins / Peroxisomal protein import / Iron uptake and transport / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Antigen processing: Ubiquitination & Proteasome degradation / AAA+ lid domain / Neutrophil degranulation / Dual incision in TC-NER / SCF(Skp2)-mediated degradation of p27/p21 / MAPK6/MAPK4 signaling / ABC-family proteins mediated transport / AUF1 (hnRNP D0) binds and destabilizes mRNA / Translesion synthesis by POLK / Translesion synthesis by POLI / Termination of translesion DNA synthesis / TNFR2 non-canonical NF-kB pathway / Negative regulation of MAPK pathway / UCH proteinases / Formation of TC-NER Pre-Incision Complex / Ub-specific processing proteases / Metalloprotease DUBs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / DNA Damage Recognition in GG-NER / Formation of Incision Complex in GG-NER / Gap-filling DNA repair synthesis and ligation in GG-NER / Dual Incision in GG-NER / Proteasome subunit alpha6 / 26S Proteasome regulatory subunit 6A / Proteasome beta 3 subunit / 26S proteasome regulatory subunit P45-like / Ubiquitin-like domain superfamily / Proteasome alpha-subunit, N-terminal domain / JAB1/MPN/MOV34 metalloenzyme domain / Ubiquitin domain / Proteasome, subunit alpha/beta / Ribosomal protein L40e / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit beta 4 / Ubiquitin conserved site / Ubiquitin / Proteasome alpha-type subunit / Proteasome B-type subunit / Proteasome beta subunit, C-terminal / Rpn11/EIF3F, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Nucleophile aminohydrolases, N-terminal
Function and homology information
Specimen sourceSaccharomyces cerevisiae S288c (yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.17 Å resolution
Authorsde la Pena, A.H. / Goodall, E.A. / Gates, S.N. / Lander, G.C. / Martin, A.
CitationJournal: 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 / A Martin
Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and ...The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric 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-EM 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+ motor to induce conformational changes and propel the substrate through the central pore.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 15, 2018 / Release: Oct 17, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 17, 2018Structure modelrepositoryInitial release
1.1Oct 24, 2018Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Assembly

Deposited unit
1: Proteasome subunit beta type-1
2: Proteasome subunit beta type-2
3: Proteasome subunit beta type-3
4: Proteasome subunit beta type-4
5: Proteasome subunit beta type-5
6: Proteasome subunit beta type-6
7: Proteasome subunit beta type-7
A: Proteasome subunit alpha type-1
B: Proteasome subunit alpha type-2
C: Proteasome subunit alpha type-3
D: Proteasome subunit alpha type-4
E: Proteasome subunit alpha type-5
F: Proteasome subunit alpha type-6
G: Probable proteasome subunit alpha type-7
H: 26S proteasome regulatory subunit 7 homolog
I: 26S proteasome regulatory subunit 4 homolog
J: 26S proteasome regulatory subunit 8 homolog
K: 26S proteasome regulatory subunit 6B homolog
L: 26S proteasome subunit RPT4
M: 26S proteasome regulatory subunit 6A
n: Proteasome subunit beta type-7
r: Ubiquitin carboxyl-terminal hydrolase RPN11
s: Model substrate polypeptide
u: Ubiquitin-60S ribosomal protein L40
hetero molecules


Theoretical massNumber of molelcules
Total (without water)733,21530
Polyers730,33224
Non-polymers2,8836
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, purification of holoenzyme, microscopy, EM density
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)94290
ΔGint (kcal/M)-439
Surface area (Å2)253890

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Components

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Proteasome subunit beta type- ... , 8 types, 8 molecules 1234567n

#1: Protein/peptide Proteasome subunit beta type-1 / PSMB1 / Macropain subunit PRE3 / Multicatalytic endopeptidase complex subunit PRE3 / Proteasome component PRE3 / Proteinase YSCE subunit PRE3


Mass: 23573.604 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE3, YJL001W, J1407 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P38624, proteasome endopeptidase complex
#2: Protein/peptide Proteasome subunit beta type-2 / PSMB2 / Macropain subunit PUP1 / Multicatalytic endopeptidase complex subunit PUP1 / Proteasome component PUP1 / Proteinase YSCE subunit PUP1


Mass: 28299.889 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP1, YOR157C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P25043, proteasome endopeptidase complex
#3: Protein/peptide Proteasome subunit beta type-3 / PSMB3 / Macropain subunit PUP3 / Multicatalytic endopeptidase complex subunit PUP3 / Proteasome component PUP3


Mass: 22627.842 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP3, YER094C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P25451, proteasome endopeptidase complex
#4: Protein/peptide Proteasome subunit beta type-4 / PSMB4 / Macropain subunit C11 / Multicatalytic endopeptidase complex subunit C11 / Proteasome component C11 / Proteinase YSCE subunit 11


Mass: 22545.676 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE1, YER012W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P22141, proteasome endopeptidase complex
#5: Protein/peptide Proteasome subunit beta type-5 / PSMB5 / Macropain subunit PRE2 / Multicatalytic endopeptidase complex subunit PRE2 / Proteasome component PRE2 / Proteinase YSCE subunit PRE2


Mass: 31670.539 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE2, DOA3, PRG1, YPR103W, P8283.10 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P30656, proteasome endopeptidase complex
#6: Protein/peptide Proteasome subunit beta type-6 / / Multicatalytic endopeptidase complex subunit C5 / Proteasome component C5


Mass: 26905.076 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE7, PRS3, PTS1, YBL041W, YBL0407 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23724, proteasome endopeptidase complex
#7: Protein/peptide Proteasome subunit beta type-7 / / Macropain subunit PRE4 / Multicatalytic endopeptidase complex subunit PRE4 / Proteasome component PRE4 / Proteinase YSCE subunit PRE4


Mass: 29471.289 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE4, YFR050C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P30657, proteasome endopeptidase complex
#21: Protein/peptide Proteasome subunit beta type-7 /


Mass: 1789.039 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: PSB7 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40

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Proteasome subunit alpha type- ... , 6 types, 6 molecules ABCDEF

#8: Protein/peptide Proteasome subunit alpha type-1 / / Macropain subunit C7-alpha / Multicatalytic endopeptidase complex C7 / Proteasome component C7-alpha / Proteasome component Y8 / Proteinase YSCE subunit 7 / SCL1 suppressor protein


Mass: 28033.830 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: SCL1, PRC2, PRS2, YGL011C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P21243, proteasome endopeptidase complex
#9: Protein/peptide Proteasome subunit alpha type-2 / / Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / Proteinase YSCE subunit 7


Mass: 27191.828 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE8, PRS4, YML092C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23639, proteasome endopeptidase complex
#10: Protein/peptide Proteasome subunit alpha type-3 / / Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 / Proteinase YSCE subunit 13


Mass: 28748.230 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE9, PRS5, YGR135W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23638, proteasome endopeptidase complex
#11: Protein/peptide Proteasome subunit alpha type-4 / / Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component PRE6 / Proteinase YSCE subunit PRE6


Mass: 28478.111 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE6, YOL038W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P40303, proteasome endopeptidase complex
#12: Protein/peptide Proteasome subunit alpha type-5 / / Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component PUP2 / Proteinase YSCE subunit PUP2


Mass: 28649.086 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP2, DOA5, YGR253C, G9155 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P32379, proteasome endopeptidase complex
#13: Protein/peptide Proteasome subunit alpha type-6 / / Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component PRE5 / Proteinase YSCE subunit PRE5


Mass: 25634.000 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE5, YMR314W, YM9924.06 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P40302, proteasome endopeptidase complex

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Protein/peptide , 5 types, 5 molecules GLrsu

#14: Protein/peptide Probable proteasome subunit alpha type-7 / Macropain subunit C1 / Multicatalytic endopeptidase complex subunit C1 / Proteasome component C1 / Proteinase YSCE subunit 1


Mass: 31575.068 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE10, PRC1, PRS1, YOR362C, O6650 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P21242, proteasome endopeptidase complex
#19: Protein/peptide 26S proteasome subunit RPT4 / 26S protease subunit SUG2 / Proteasomal cap subunit


Mass: 49480.137 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT4, CRL13, PCS1, SUG2, YOR259C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P53549
#22: Protein/peptide Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome regulatory subunit RPN11 / Protein MPR1


Mass: 34442.281 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPN11, MPR1, YFR004W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P43588, ubiquitinyl hydrolase 1
#23: Protein/peptide Model substrate polypeptide


Mass: 4068.414 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21
#24: Protein/peptide Ubiquitin-60S ribosomal protein L40


Mass: 14583.077 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPL40A, UBI1, YIL148W / Production host: Escherichia coli B (bacteria) / References: UniProt: P0CH08

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26S proteasome regulatory subunit ... , 5 types, 5 molecules HIJKM

#15: Protein/peptide 26S proteasome regulatory subunit 7 homolog / Protein CIM5 / Tat-binding homolog 3


Mass: 52054.891 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT1, CIM5, YTA3, YKL145W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33299
#16: Protein/peptide 26S proteasome regulatory subunit 4 homolog / Tat-binding homolog 5


Mass: 48898.160 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT2, YHS4, YTA5, YDL007W, D2920 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P40327
#17: Protein/peptide 26S proteasome regulatory subunit 8 homolog / Protein CIM3 / Protein SUG1 / Tat-binding protein TBY1


Mass: 45342.742 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT6, CIM3, CRL3, SUG1, TBPY, TBY1, YGL048C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: Q01939
#18: Protein/peptide 26S proteasome regulatory subunit 6B homolog / Protein YNT1 / Tat-binding homolog 2


Mass: 47953.676 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT3, YNT1, YTA2, YDR394W, D9509.14 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33298
#20: Protein/peptide 26S proteasome regulatory subunit 6A / Tat-binding protein homolog 1 / TBP-1


Mass: 48315.727 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT5, YTA1, YOR117W, O3258, YOR3258W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33297

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Non-polymers , 2 types, 6 molecules

#25: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM
#26: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameDetailsEntity IDParent IDSource
1Substrate-engaged 26S proteasome in the 4D stateYeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 240MULTIPLE SOURCES
2Proteasome1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 231RECOMBINANT
3substrate241RECOMBINANT
Source (natural)
IDEntity assembly IDNcbi tax IDOrganismStrain
12559292Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)ATCC 204508 / S288c
239606Homo sapiens (human)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismStrain
92580240Saccharomyces cerevisiae W303 (yeast)
103511693Escherichia coli BL21 (bacteria)BL21
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer ID
120 mMHEPES1
225 mMNaCl1
325 mMKCl1
410 mMMgCl21
51 mMTCEP1
65 mMATP1
70.05 % (w/v)Nonidet P-401
86 mMortho-phenanthroline1
SpecimenConc.: 25 mg/ml
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
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 kelvins
Details: specimens were manually blotted with Whatman #1 filter paper

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS / Details: images were acquired in nanoprobe mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 / 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 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.25 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 11656
Image scansSampling size: 5 microns / Width: 3710 / Height: 3838 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4MotionCorr22CTF correction
5RELION2.1CTF correction
6CTFFIND4CTF correction
7GctfCTF correction
10UCSF Chimera1.12model fitting
11PHENIX1.11-2580model fitting
13RELION2.1initial Euler assignment
14RELION2.1final Euler assignment
16RELION2.13D reconstruction
17PHENIX1.11-2580model refinement
Image processingDetails: Camera was operated in counting mode
CTF correctionDetails: CTF correction was performed by Relion during reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Particles were selected using the Relion template-based particle picker
Number of particles selected: 579361
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 48335 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 5MPC

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