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- EMDB-5741: Single-particle Electron Microscopy Structure of the TRAPPIII Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5741
TitleSingle-particle Electron Microscopy Structure of the TRAPPIII Complex
Map dataReconstruction of yeast TRAPPIII complex, filtered to 22 Angstrom resolution
Sample
  • Sample: Recombinant TRAPPIII complex
  • Protein or peptide: transport protein particle III
Keywordsautophagy
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsTan D / Cai Y / Wang J / Zhang J / Menon S / Chou H-T / Ferro-Novick S / Reinisch KM / Walz T
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway.
Authors: Dongyan Tan / Yiying Cai / Juan Wang / Jinzhong Zhang / Shekar Menon / Hui-Ting Chou / Susan Ferro-Novick / Karin M Reinisch / Thomas Walz /
Abstract: The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 ...The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.
History
DepositionAug 12, 2013-
Header (metadata) releaseSep 4, 2013-
Map releaseNov 13, 2013-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.507
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.507
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5741_1.tif

Downloads & links

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Map

FileDownload / File: emd_5741.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast TRAPPIII complex, filtered to 22 Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.48 Å/pix.
x 80 pix.
= 358.4 Å		4.48 Å/pix.
x 80 pix.
= 358.4 Å		4.48 Å/pix.
x 80 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.507 / Movie #1: 0.507
Minimum - Maximum-0.61211646 - 2.31793046
Average (Standard dev.)0.00571768 (±0.11935551)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.484.484.48
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.6122.3180.006

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Supplemental data

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Sample components

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Entire : Recombinant TRAPPIII complex

EntireName: Recombinant TRAPPIII complex
Components
  • Sample: Recombinant TRAPPIII complex
  • Protein or peptide: transport protein particle III

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Supramolecule #1000: Recombinant TRAPPIII complex

SupramoleculeName: Recombinant TRAPPIII complex / type: sample / ID: 1000 / Oligomeric state: oligomer / Number unique components: 1
Molecular weightExperimental: 254 KDa / Theoretical: 254 KDa

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Macromolecule #1: transport protein particle III

MacromoleculeName: transport protein particle III / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 254 KDa / Theoretical: 254 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pETDuet

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 8 / Details: 20mM Tris-HCl, 300 mM NaCl, 1mM DTT
StainingType: NEGATIVE
Details: Grids with adsorbed protein stained with 0.75% uranyl format for 20 seconds
GridDetails: 200 mesh copper grids with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Detailslow dose setting
DateDec 20, 2010
Image recordingCategory: FILM / Film or detector model: GENERIC IMAGE PLATES / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 133 / Average electron dose: 10 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 67000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 67000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 60

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Image processing

DetailsRandom Conical Tilt reconstruction using Spider scripts
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider
Details: Final maps were calculated from a dataset of two class-averages
Number images used: 1120
Final two d classificationNumber classes: 20

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Atomic model buiding 1

Initial modelPDB ID:

2j3t

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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