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- EMDB-26247: In situ architecture of VPS13C -

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Basic information

Entry
Database: EMDB / ID: EMD-26247
TitleIn situ architecture of VPS13C
Map data
Sample
  • Cell: full-length human VPS13C bridging the two adjacent membranes
KeywordsLipid transfer / LIPID TRANSPORT
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 47.0 Å
AuthorsCai S / De Camilli P
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA018343 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5R01AI152421 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: In situ architecture of the lipid transport protein VPS13C at ER-lysosome membrane contacts.
Authors: Shujun Cai / Yumei Wu / Andrés Guillén-Samander / William Hancock-Cerutti / Jun Liu / Pietro De Camilli /
Abstract: VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct ...VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson's disease-linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER-endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.
History
DepositionFeb 17, 2022-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26247.png

Downloads & links

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Map

FileDownload / File: emd_26247.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
13.54 Å/pix.
x 64 pix.
= 866.304 Å		13.54 Å/pix.
x 64 pix.
= 866.304 Å		13.54 Å/pix.
x 64 pix.
= 866.304 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 13.536 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.3
Minimum - Maximum-0.5152203 - 2.0491447
Average (Standard dev.)0.00045576657 (±0.084811926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 866.304 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26247_additional_1.map
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Additional map: #2

Fileemd_26247_additional_2.map
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Sample components

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Entire : full-length human VPS13C bridging the two adjacent membranes

EntireName: full-length human VPS13C bridging the two adjacent membranes
Components
  • Cell: full-length human VPS13C bridging the two adjacent membranes

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Supramolecule #1: full-length human VPS13C bridging the two adjacent membranes

SupramoleculeName: full-length human VPS13C bridging the two adjacent membranes
type: cell / ID: 1 / Parent: 0
Details: Subtomogram-average density maps showing a full-length VPS13C rod bridging the two adjacent membranes. C100 symmetry was applied to enhance signal to noise ratio.
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -0.006 µm / Nominal defocus min: -0.001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 47.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PROTOMO / Number subtomograms used: 570
ExtractionNumber tomograms: 20 / Number images used: 570
Final angle assignmentType: NOT APPLICABLE

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