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- PDB-7kmt: Structure of the yeast TRAPPIII-Ypt1(Rab1) complex -

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Basic information

Entry
Database: PDB / ID: 7kmt
TitleStructure of the yeast TRAPPIII-Ypt1(Rab1) complex
Components
  • (Trafficking protein particle complex subunit ...) x 6
  • GTP-binding protein YPT1
  • Trafficking protein particle complex III-specific subunit 85
KeywordsPROTEIN TRANSPORT / GTPase / GEF / ER / Golgi / Autophagy
Function / homology
Function and homology information


Cvt vesicle assembly / pre-mRNA catabolic process / autophagy of peroxisome / Golgi Cisternae Pericentriolar Stack Reorganization / Golgi vesicle docking / regulation of endoplasmic reticulum unfolded protein response / Golgi vesicle budding / RAB geranylgeranylation / TRAPPI protein complex / SNARE complex disassembly ...Cvt vesicle assembly / pre-mRNA catabolic process / autophagy of peroxisome / Golgi Cisternae Pericentriolar Stack Reorganization / Golgi vesicle docking / regulation of endoplasmic reticulum unfolded protein response / Golgi vesicle budding / RAB geranylgeranylation / TRAPPI protein complex / SNARE complex disassembly / RAB GEFs exchange GTP for GDP on RABs / TRAPPII protein complex / TRAPPIII protein complex / TRAPP complex / early endosome to Golgi transport / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / COPII-coated vesicle budding / COPII-mediated vesicle transport / cytoplasm to vacuole targeting by the Cvt pathway / intra-Golgi vesicle-mediated transport / protein localization to phagophore assembly site / piecemeal microautophagy of the nucleus / phagophore assembly site membrane / Golgi stack / cis-Golgi network / protein-containing complex localization / endocytic recycling / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / phagophore assembly site / reticulophagy / retrograde transport, endosome to Golgi / cis-Golgi network membrane / SNARE complex assembly / sporulation resulting in formation of a cellular spore / positive regulation of macroautophagy / autophagosome assembly / chromosome organization / endoplasmic reticulum to Golgi vesicle-mediated transport / endomembrane system / Neutrophil degranulation / SNARE binding / meiotic cell cycle / macroautophagy / intracellular protein transport / trans-Golgi network / cytoplasmic vesicle / protein-containing complex assembly / Golgi membrane / GTPase activity / GTP binding / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / mitochondrion / nucleus / cytosol / cytoplasm
Similarity search - Function
TRAPP III complex, Trs85 / ER-Golgi trafficking TRAPP I complex 85 kDa subunit / Trafficking protein particle complex subunit 2 / Sedlin, N-terminal conserved region / Trafficking protein particle complex subunit / Sybindin-like family / Sybindin-like family / TRAPP I complex, subunit 5 / TRAPP complex, Trs33 subunit / Bet3 family ...TRAPP III complex, Trs85 / ER-Golgi trafficking TRAPP I complex 85 kDa subunit / Trafficking protein particle complex subunit 2 / Sedlin, N-terminal conserved region / Trafficking protein particle complex subunit / Sybindin-like family / Sybindin-like family / TRAPP I complex, subunit 5 / TRAPP complex, Trs33 subunit / Bet3 family / Transport protein particle (TRAPP) component / Transport protein particle (TRAPP) component / NO signalling/Golgi transport ligand-binding domain superfamily / Longin-like domain superfamily / small GTPase Rab1 family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PALMITIC ACID / GTP-binding protein YPT1 / Trafficking protein particle complex subunit BET3 / Trafficking protein particle complex subunit 20 / Trafficking protein particle complex III-specific subunit 85 / Trafficking protein particle complex subunit 31 / Trafficking protein particle complex subunit BET5 / Trafficking protein particle complex subunit 23 / Trafficking protein particle complex subunit 33
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsJoiner, A.M.N. / Phillips, B.P. / Miller, E.A. / Fromme, J.C.
Funding support United States, United Kingdom, 10items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM116942 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM097272 United States
National Institutes of Health/National Institute on Minority Health and Health Disparities (NIH/NIMHD)R01HD095296 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124559 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124559 United States
National Science Foundation (NSF, United States)DBI-1661380 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
Medical Research Council (MRC, United Kingdom)MRC_UP_1201/10 United Kingdom
National Science Foundation (NSF, United States)DGE-1650441 United States
CitationJournal: EMBO J / Year: 2021
Title: Structural basis of TRAPPIII-mediated Rab1 activation.
Authors: Aaron Mn Joiner / Ben P Phillips / Kumar Yugandhar / Ethan J Sanford / Marcus B Smolka / Haiyuan Yu / Elizabeth A Miller / J Christopher Fromme /
Abstract: The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII ...The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.
History
DepositionNov 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 23, 2021Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-22928
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Structure viewerMolecule:
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Assembly

Deposited unit
H: Trafficking protein particle complex subunit 23
J: Trafficking protein particle complex subunit 31
G: Trafficking protein particle complex subunit BET5
I: Trafficking protein particle complex subunit BET3
F: Trafficking protein particle complex subunit BET3
E: Trafficking protein particle complex subunit 33
K: Trafficking protein particle complex subunit 20
A: GTP-binding protein YPT1
B: Trafficking protein particle complex III-specific subunit 85
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,51411
Polymers276,0019
Non-polymers5132
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, This is a well-established complex.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31460 Å2
ΔGint-162 kcal/mol
Surface area75060 Å2

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Components

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Trafficking protein particle complex subunit ... , 6 types, 7 molecules HJGIFEK

#1: Protein Trafficking protein particle complex subunit 23 / TRAPP subunit 23 / Transport protein particle 23 kDa subunit


Mass: 24889.262 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TRS23, YDR246W, YD8419.13 / Production host: Escherichia coli (E. coli) / References: UniProt: Q03784
#2: Protein Trafficking protein particle complex subunit 31 / TRAPP subunit 31 / Transport protein particle 31 kDa subunit


Mass: 31755.689 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TRS31, YDR472W / Production host: Escherichia coli (E. coli) / References: UniProt: Q03337
#3: Protein Trafficking protein particle complex subunit BET5 / TRAPP subunit BET5 / Transport protein particle 18 kDa subunit


Mass: 18453.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: BET5, YML077W / Production host: Escherichia coli (E. coli) / References: UniProt: Q03630
#4: Protein Trafficking protein particle complex subunit BET3 / TRAPP subunit BET3 / Transport protein particle 22 kDa subunit


Mass: 22152.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: BET3, YKR068C / Production host: Escherichia coli (E. coli) / References: UniProt: P36149
#5: Protein Trafficking protein particle complex subunit 33 / TRAPP subunit 33 / Transport protein particle 33 kDa subunit


Mass: 30786.176 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TRS33, YOR115C, O3251, YOR3251C / Production host: Escherichia coli (E. coli) / References: UniProt: Q99394
#6: Protein Trafficking protein particle complex subunit 20 / TRAPP subunit 20 / Transport protein particle 20 kDa subunit


Mass: 19721.154 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TRS20, YBR254C, YBR1722 / Production host: Escherichia coli (E. coli) / References: UniProt: P38334

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Protein , 2 types, 2 molecules AB

#7: Protein GTP-binding protein YPT1 / Protein YP2 / Rab GTPase YPT1 / Transport GTPase YPT1


Mass: 23240.227 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YPT1, YP2, YFL038C / Production host: Escherichia coli (E. coli) / References: UniProt: P01123
#8: Protein Trafficking protein particle complex III-specific subunit 85 / TRAPP III-specific subunit 85 / Muddled meiosis protein 1 / Sporulation protein GSG1 / Transport ...TRAPP III-specific subunit 85 / Muddled meiosis protein 1 / Sporulation protein GSG1 / Transport protein particle 85 kDa subunit


Mass: 82850.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TRS85, GSG1, MUM1, YDR108W, YD9727.04 / Production host: Escherichia coli (E. coli) / References: UniProt: P46944

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Non-polymers , 1 types, 2 molecules

#9: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H32O2

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TRAPPIII-Ypt1 complex / Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
110 mMTris1
2350 mMsodium chloride1
31 mMDTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Either 0.02% Tween-20 or 0.025% amphipol A8-35 was added before application of the sample to the grid.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company /
Model: Titan Krios / Image courtesy: FEI Company
EM imaging
IDAccelerating voltage (kV)Electron sourceIllumination modeModelModeSpecimen-ID
1200FIELD EMISSION GUNFLOOD BEAMFEI TALOS ARCTICABRIGHT FIELDBright-field microscopy1
2300FIELD EMISSION GUNFLOOD BEAMFEI TITAN KRIOSBRIGHT FIELDBright-field microscopy1
3300FIELD EMISSION GUNFLOOD BEAMFEI TITAN KRIOSBRIGHT FIELDBright-field microscopy1
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelDetector mode
1150GATAN K3 BIOQUANTUM (6k x 4k)
2220FEI FALCON III (4k x 4k)COUNTING
3350GATAN K2 SUMMIT (4k x 4k)SUPER-RESOLUTION
EM imaging optics
Energyfilter nameIDImaging-IDEnergyfilter slit width (eV)
GIF Bioquantum1120
22
GIF Quantum LS3320

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Processing

EM software
IDNameVersionCategoryImaging-ID
1RELION3.1particle selection
2SerialEMimage acquisition1
4RELION3.1CTF correction
7Cootmodel fitting
8ISOLDEmodel fitting
9UCSF Chimeramodel fitting
11EPUimage acquisition2
12SerialEMimage acquisition3
13RELION3.1initial Euler assignment
14RELION3.1final Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
17PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69315 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
13CUE

3cue

1
22C0J

2c0j

1
32J3W

2j3w

1

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