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- EMDB-8900: Negative stain reconstruction of human autophagy associated prote... -

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Basic information

Entry
Database: EMDB / ID: EMD-8900
TitleNegative stain reconstruction of human autophagy associated protein, ATG2A
Map dataNegative stain reconstruction of human ATG2A protein
Sample
  • Complex: Human ATG2A protein
Function / homology
Function and homology information


organelle membrane contact site / lipid transfer activity / positive regulation of autophagosome assembly / nucleophagy / piecemeal microautophagy of the nucleus / phagophore assembly site membrane / phosphatidylinositol-3-phosphate binding / phagophore assembly site / reticulophagy / autophagosome assembly ...organelle membrane contact site / lipid transfer activity / positive regulation of autophagosome assembly / nucleophagy / piecemeal microautophagy of the nucleus / phagophore assembly site membrane / phosphatidylinositol-3-phosphate binding / phagophore assembly site / reticulophagy / autophagosome assembly / lipid droplet / endoplasmic reticulum membrane
Similarity search - Function
Autophagy-related protein 2/VPS13, C-terminal / Autophagy-related protein 2 / ATG2/VPS13, C terminal domain / Vacuolar protein sorting-associated protein 13-like, N-terminal domain / VPS13-like, N-terminal
Similarity search - Domain/homology
Autophagy-related protein 2 homolog A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 29.4 Å
AuthorsChowdhury S / Otomo C / Leitner A / Ohashi K / Aebersold R / Lander GC / Otomo T
Funding support United States, European Union, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM092740, DP2EB020402 United States
European Research Council (ERC)670821European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex.
Authors: Saikat Chowdhury / Chinatsu Otomo / Alexander Leitner / Kazuto Ohashi / Ruedi Aebersold / Gabriel C Lander / Takanori Otomo /
Abstract: Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for ...Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.
History
DepositionAug 21, 2017-
Header (metadata) releaseSep 6, 2017-
Map releaseSep 6, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8900.png

Downloads & links

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Map

FileDownload / File: emd_8900.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of human ATG2A protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 96 pix.
= 393.6 Å		4.1 Å/pix.
x 96 pix.
= 393.6 Å		4.1 Å/pix.
x 96 pix.
= 393.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.2 / Movie #1: 5.2
Minimum - Maximum-5.7097316 - 19.625645
Average (Standard dev.)0.057370227 (±0.7065861)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 393.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-5.71019.6260.057

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Supplemental data

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Additional map: Unsharpened negative stain EM map of human ATG2A protein

Fileemd_8900_additional.map
AnnotationUnsharpened negative stain EM map of human ATG2A protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human ATG2A protein

EntireName: Human ATG2A protein
Components
  • Complex: Human ATG2A protein

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Supramolecule #1: Human ATG2A protein

SupramoleculeName: Human ATG2A protein / type: complex / ID: 1 / Parent: 0 / Details: Full length protein
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: 9
Molecular weightTheoretical: 200 KDa

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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

Concentration0.01 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: Uranyl Formate
GridModel: Maxtaform / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.62 µm / Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 273.15 K / Max: 298.15 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Number grids imaged: 1 / Number real images: 590 / Average exposure time: 0.42 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 37021
CTF correctionSoftware - Name: RELION (ver. 1.4)
Startup modelType of model: EMDB MAP
EMDB ID:

8899


Details: A negative stain reconstruction of human ATG2-WIPI4 complex was low pass filtered to 60 Angstrom and used as initial model.
Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 29.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 6386

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