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- EMDB-11212: Cryo-EM structure of ESCRT-III helical Vps24 filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-11212
TitleCryo-EM structure of ESCRT-III helical Vps24 filaments
Map dataSharpened cryo EM density
Sample
  • Complex: Doubled-stranded helical filament assembly of Vps24
    • Protein or peptide: Vacuolar protein-sorting-associated protein 24Vacuole
Function / homology
Function and homology information


Endosomal Sorting Complex Required For Transport (ESCRT) / intralumenal vesicle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Macroautophagy / ATP export / ESCRT III complex / endosome transport via multivesicular body sorting pathway / late endosome to vacuole transport / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body ...Endosomal Sorting Complex Required For Transport (ESCRT) / intralumenal vesicle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Macroautophagy / ATP export / ESCRT III complex / endosome transport via multivesicular body sorting pathway / late endosome to vacuole transport / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body / protein transport / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Vacuolar protein-sorting-associated protein 24
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHuber ST / Mostafavi S / Mortensen SA / Sachse C
CitationJournal: Sci Adv / Year: 2020
Title: Structure and assembly of ESCRT-III helical Vps24 filaments.
Authors: Stefan T Huber / Siavash Mostafavi / Simon A Mortensen / Carsten Sachse /
Abstract: ESCRT-III proteins mediate a range of cellular membrane remodeling activities such as multivesicular body biogenesis, cytokinesis, and viral release. Critical to these processes is the assembly of ...ESCRT-III proteins mediate a range of cellular membrane remodeling activities such as multivesicular body biogenesis, cytokinesis, and viral release. Critical to these processes is the assembly of ESCRT-III subunits into polymeric structures. In this study, we determined the cryo-EM structure of a helical assembly of Vps24 at 3.2-Å resolution and found that Vps24 adopts an elongated open conformation. Vps24 forms a domain-swapped dimer extended into protofilaments that associate into a double-stranded apolar filament. We demonstrate that, upon binding negatively charged lipids, Vps24 homopolymer filaments undergo partial disassembly into shorter filament fragments and oligomers. Upon the addition of Vps24, Vps2, and Snf7, liposomes are deformed into neck and tubular structures by an ESCRT-III heteropolymer coat. The filamentous Vps24 homopolymer assembly structure and interaction studies reveal how Vps24 could introduce unique geometric properties to mixed-type ESCRT-III heteropolymers and contribute to the process of membrane scission events.
History
DepositionJun 20, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateSep 16, 2020-
Current statusSep 16, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zh3
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zh3
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11212.png

Downloads & links

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Map

FileDownload / File: emd_11212.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo EM density
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.22486193 - 0.38453808
Average (Standard dev.)0.001851589 (±0.015475936)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2250.3850.002

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Supplemental data

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Mask #1

Fileemd_11212_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo EM density

Fileemd_11212_additional.map
AnnotationUnsharpened cryo EM density
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_11212_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_11212_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Doubled-stranded helical filament assembly of Vps24

EntireName: Doubled-stranded helical filament assembly of Vps24
Components
  • Complex: Doubled-stranded helical filament assembly of Vps24
    • Protein or peptide: Vacuolar protein-sorting-associated protein 24Vacuole

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Supramolecule #1: Doubled-stranded helical filament assembly of Vps24

SupramoleculeName: Doubled-stranded helical filament assembly of Vps24 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Vacuolar protein-sorting-associated protein 24

MacromoleculeName: Vacuolar protein-sorting-associated protein 24 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 26.562174 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMDYIKKA IWGPDPKEQQ RRIRSVLRKN GRNIEKSLRE LTVLQNKTQQ LIKKSAKKND VRTVRLYAKE LYQINKQYDR MYTSRAQLD SVRMKIDEAI RMNTLSNQMA DSAGLMREVN SLVRLPQLRN TMIELEKELM KSGIISEMVD DTMESVGDVG E EMDEAVDE ...String:
GSHMDYIKKA IWGPDPKEQQ RRIRSVLRKN GRNIEKSLRE LTVLQNKTQQ LIKKSAKKND VRTVRLYAKE LYQINKQYDR MYTSRAQLD SVRMKIDEAI RMNTLSNQMA DSAGLMREVN SLVRLPQLRN TMIELEKELM KSGIISEMVD DTMESVGDVG E EMDEAVDE EVNKIVEQYT NEKFKNVDQV PTVELAANEE EQEIPDEKVD EEADRMVNEM RERLRALQN

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.9 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
20.0 mMTrisTris
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was concentrated to 5 mg/mL, incubated in the refridgerator overnight, ultracentrifugated, and resuspended to a final concentration of 0.9 mg/mL for cryo-EM

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number real images: 3257 / Average electron dose: 1.0 e/Å2
Details: 1320 images were manually selected for further processing
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 524155 / Software - Name: RELION (ver. 2.1) / Details: one segment every 24.7 Angstrom
CTF correctionSoftware - Name: RELION (ver. 2.1)
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 62
Applied symmetry - Helical parameters - Δz: 25.18 Å
Applied symmetry - Helical parameters - Δ&Phi: -32.47 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 313554
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3frt


Chain - Chain ID: A
RefinementSpace: REAL / Overall B value: 126
Output model

PDB-6zh3:
Cryo-EM structure of ESCRT-III helical Vps24 filaments

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