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- EMDB-11376: Subtomogram averaging of the OSBP construct NPHFFAT. -

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Basic information

Entry
Database: EMDB / ID: EMD-11376
TitleSubtomogram averaging of the OSBP construct NPHFFAT.
Map dataNPHFFAT in galactocerebroside tubes containing PI4P
Sample
  • Complex: Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Binding Protein
    • Other: N-PH-FFAT (OSBP 1-407)
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 9.8 Å
Authorsde la Mora E / Dezi M / diCicco A / Bigay J / Gautier R / Manzi J / Polidori J / Castano-Diez D / Mesmin B / Antonny B / Levy D
CitationJournal: Nat Commun / Year: 2021
Title: Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites.
Authors: Eugenio de la Mora / Manuela Dezi / Aurélie Di Cicco / Joëlle Bigay / Romain Gautier / John Manzi / Joël Polidori / Daniel Castaño-Díez / Bruno Mesmin / Bruno Antonny / Daniel Lévy /
Abstract: Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is ...Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.
History
DepositionJul 10, 2020-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.511
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.511
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11376.png

Downloads & links

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Map

FileDownload / File: emd_11376.map.gz / Format: CCP4 / Size: 549.8 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNPHFFAT in galactocerebroside tubes containing PI4P
Voxel sizeX=Y=Z: 3.4 Å
Density
Contour LevelBy AUTHOR: 0.511 / Movie #1: 0.511
Minimum - Maximum-3.1721237 - 7.978864
Average (Standard dev.)-0.009864336 (±0.52696496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions525252
Spacing525252
CellA=B=C: 176.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.43.43.4
M x/y/z525252
origin x/y/z0.0000.0000.000
length x/y/z176.800176.800176.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS525252
D min/max/mean-3.1727.979-0.010

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Supplemental data

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Sample components

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Entire : Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Bi...

EntireName: Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Binding Protein
Components
  • Complex: Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Binding Protein
    • Other: N-PH-FFAT (OSBP 1-407)

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Supramolecule #1: Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Bi...

SupramoleculeName: Chimera N-PH-FFAT comprising residues 1-407 from the Oxysterol-Binding Protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 43.2 kDa/nm

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Macromolecule #1: N-PH-FFAT (OSBP 1-407)

MacromoleculeName: N-PH-FFAT (OSBP 1-407) / type: other / ID: 1
Details: Chimera N-PH-FFAT comprising residues 1-407 from human Oxysterol-Binding Protein.
Classification: other
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAATELRGVV GPGPAAIAAL GGGGAGPPVV GGGGGRGDAG PGSGAASGTV VAAAAGGPGP GAGGVAAAGP APAPPTGGSG GSGAGGSGSA REGWLFKWTN YIKGYQRRWF VLSNGLLSYY RSKAEMRHTC RGTINLATAN ITVEDSCNFI ISNGGAQTYH LKASSEVERQ ...String:
MAATELRGVV GPGPAAIAAL GGGGAGPPVV GGGGGRGDAG PGSGAASGTV VAAAAGGPGP GAGGVAAAGP APAPPTGGSG GSGAGGSGSA REGWLFKWTN YIKGYQRRWF VLSNGLLSYY RSKAEMRHTC RGTINLATAN ITVEDSCNFI ISNGGAQTYH LKASSEVERQ RWVTALELAK AKAVKMLAES DESGDEESVS QTDKTELQNT LRTLSSKVED LSTCNDLIAK HGTALQRSLS ELESLKLPAE SNEKIKQVNE RATLFRITSN AMINACRDFL MLAQTHSKKW QKSLQYERDQ RIRLEETLEQ LAKQHNHLER AFRGATVLPA NTPGNVGSGK DQCCSGKGDM SDEDDENEFF DAPEIITMPE NLGHKRTGSN ISGASSDISL DEQYKHQLEE TKKEKRTR
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
120.0 mMC2H3KO2Potassium acetate

Details: 50 mM HEPES pH 7.4 120 mM potassium acetate
GridModel: Homemade / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 21 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Average electron dose: 3.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 50 / Number images used: 15236 / Method: Subtomogram averaging / Software - Name: Dynamo (ver. 1.1.478)
CTF correctionSoftware - Name: CTFPHASEFLIP
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 15236
FSC plot (resolution estimation)

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