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

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Basic information

Entry
Database: PDB / ID: 6zh3
TitleCryo-EM structure of ESCRT-III helical Vps24 filaments
ComponentsVacuolar protein-sorting-associated protein 24Vacuole
KeywordsLIPID BINDING PROTEIN / Filament / Helical / Membrane Remodeling
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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHuber, S.T. / Mostafavi, S. / Mortensen, S.A. / 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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 26, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
B: Vacuolar protein-sorting-associated protein 24
A: Vacuolar protein-sorting-associated protein 24
C: Vacuolar protein-sorting-associated protein 24
D: Vacuolar protein-sorting-associated protein 24


Theoretical massNumber of molelcules
Total (without water)106,2494
Polymers106,2494
Non-polymers00
Water0
1
B: Vacuolar protein-sorting-associated protein 24
A: Vacuolar protein-sorting-associated protein 24
C: Vacuolar protein-sorting-associated protein 24
D: Vacuolar protein-sorting-associated protein 24
x 9


Theoretical massNumber of molelcules
Total (without water)956,23836
Polymers956,23836
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation8

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Components

#1: Protein
Vacuolar protein-sorting-associated protein 24 / Vacuole / DOA4-independent degradation protein 3 / ESCRT-III complex subunit VPS24


Mass: 26562.174 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: VPS24, DID3, YKL041W, YKL254 / Production host: Escherichia coli (E. coli) / References: UniProt: P36095

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Doubled-stranded helical filament assembly of Vps24 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMTrisTris1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample 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
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3257
Details: 1320 images were manually selected for further processing
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
4RELION2.1CTF correction
7Cootmodel fitting
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -32.47 ° / Axial rise/subunit: 25.18 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 524155 / Details: one segment every 24.7 Angstrom
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 313554 / Algorithm: BACK PROJECTION / Num. of class averages: 62 / Symmetry type: HELICAL
Atomic model buildingB value: 126 / Space: REAL
Atomic model buildingPDB-ID: 3FRT

3frt


Pdb chain-ID: A

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