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- PDB-6tza: CryoEM reconstruction of ESCRT-III filament composed of IST1 NTD ... -

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Basic information

Entry
Database: PDB / ID: 6tza
TitleCryoEM reconstruction of ESCRT-III filament composed of IST1 NTD R16E K27E double mutant
ComponentsIST1 homolog
KeywordsLIPID BINDING PROTEIN / membrane remodeling / membrane-bound protein filament / ESCRT-III
Function / homology
Function and homology information


MIT domain binding / abscission / ESCRT III complex disassembly / cytoskeleton-dependent cytokinesis / collateral sprouting / positive regulation of collateral sprouting / Sealing of the nuclear envelope (NE) by ESCRT-III / multivesicular body assembly / Flemming body / positive regulation of proteolysis ...MIT domain binding / abscission / ESCRT III complex disassembly / cytoskeleton-dependent cytokinesis / collateral sprouting / positive regulation of collateral sprouting / Sealing of the nuclear envelope (NE) by ESCRT-III / multivesicular body assembly / Flemming body / positive regulation of proteolysis / endoplasmic reticulum-Golgi intermediate compartment / establishment of protein localization / azurophil granule lumen / protein localization / protein transport / nuclear envelope / midbody / cadherin binding / protein domain specific binding / cell division / intracellular membrane-bounded organelle / centrosome / Neutrophil degranulation / protein-containing complex binding / chromatin / extracellular exosome / extracellular region / identical protein binding / cytosol
Similarity search - Function
Vacuolar protein sorting-associated protein Ist1 / Vacuolar protein sorting-associated protein IST1-like / Regulator of Vps4 activity in the MVB pathway
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsNguyen, H.C. / Frost, A.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50 AI150464 United States
National Institutes of Health/Office of the DirectorS10OD020054 United States
National Institutes of Health/Office of the DirectorS10OD021741 United States
National Institutes of Health/Office of the Director1S10OD021596-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM110772-01 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Membrane constriction and thinning by sequential ESCRT-III polymerization.
Authors: Henry C Nguyen / Nathaniel Talledge / John McCullough / Abhimanyu Sharma / Frank R Moss / Janet H Iwasa / Michael D Vershinin / Wesley I Sundquist / Adam Frost /
Abstract: The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; ...The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.
History
DepositionAug 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 22, 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
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure viewerMolecule:
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Assembly

Deposited unit
A: IST1 homolog
B: IST1 homolog
C: IST1 homolog
D: IST1 homolog
E: IST1 homolog
F: IST1 homolog
G: IST1 homolog
H: IST1 homolog
I: IST1 homolog
J: IST1 homolog
K: IST1 homolog
L: IST1 homolog
M: IST1 homolog
N: IST1 homolog


Theoretical massNumber of molelcules
Total (without water)301,64614
Polymers301,64614
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 14 / Rise per n subunits: 3 Å / Rotation per n subunits: 26.85 °)

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Components

#1: Protein
IST1 homolog / hIST1 / Putative MAPK-activating protein PM28


Mass: 21546.135 Da / Num. of mol.: 14 / Fragment: N-terminal domain (UNP residues 1-189) / Mutation: R16E K27E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IST1, KIAA0174 / Production host: Escherichia coli (E. coli) / References: UniProt: P53990

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: ESCRT-III filament composed of N-terminal domain of the IST1 R16E K27E double mutant
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 292 K
Details: Grids were blotted with Whatman No. 1 filter paper for 4-8 seconds with a 0 mm offset at 19C and 100 percent humidity before plunging into liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 53 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
9PHENIXmodel refinement
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 26.85 ° / Axial rise/subunit: 3 Å / Axial symmetry: C1
3D reconstructionResolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4556 / Symmetry type: HELICAL

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