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- EMDB-20591: CryoEM reconstruction of ESCRT-III filament composed of IST1 NTD ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20591
TitleCryoEM reconstruction of ESCRT-III filament composed of IST1 NTD R16E K27E double mutant
Map dataRELION postprocessed map with helical symmetry applied to the entire map
Sample
  • Complex: ESCRT-III filament composed of N-terminal domain of the IST1 R16E K27E double mutant
    • Protein or peptide: IST1 homolog
Keywordsmembrane remodeling / membrane-bound protein filament / ESCRT-III / LIPID BINDING PROTEIN
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)
Methodhelical reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsNguyen HC / Frost A
Funding support United States, 5 items
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, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseApr 8, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
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  • Surface view with fitted model
  • Atomic models: PDB-6tza
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tza
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20591.png

Downloads & links

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Map

FileDownload / File: emd_20591.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION postprocessed map with helical symmetry applied to the entire map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 320 pix.
= 394.88 Å		1.23 Å/pix.
x 320 pix.
= 394.88 Å		1.23 Å/pix.
x 320 pix.
= 394.88 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.234 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.011055191 - 0.029239483
Average (Standard dev.)0.00018896039 (±0.0028973618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 394.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2341.2341.234
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z394.880394.880394.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0110.0290.000

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Supplemental data

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Mask #1

Fileemd_20591_msk_1.map
Projections & Slices
AxesZYX

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Additional map: RELION Refine3D filtered map with helical symmetry imposed...

Fileemd_20591_additional.map
AnnotationRELION Refine3D filtered map with helical symmetry imposed on the central 30%
Projections & Slices
AxesZYX

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Half map: RELION Refine3D unfiltered half map1 with helical symmetry...

Fileemd_20591_half_map_1.map
AnnotationRELION Refine3D unfiltered half map1 with helical symmetry imposed on the central 30%
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION Refine3D unfiltered half map2 with helical symmetry...

Fileemd_20591_half_map_2.map
AnnotationRELION Refine3D unfiltered half map2 with helical symmetry imposed on the central 30%
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ESCRT-III filament composed of N-terminal domain of the IST1 R16E...

EntireName: ESCRT-III filament composed of N-terminal domain of the IST1 R16E K27E double mutant
Components
  • Complex: ESCRT-III filament composed of N-terminal domain of the IST1 R16E K27E double mutant
    • Protein or peptide: IST1 homolog

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Supramolecule #1: ESCRT-III filament composed of N-terminal domain of the IST1 R16E...

SupramoleculeName: ESCRT-III filament composed of N-terminal domain of the IST1 R16E K27E double mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: IST1 homolog

MacromoleculeName: IST1 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.546135 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLGSGFKAER LRVNLELVIN RLKLLEEKKT ELAQKARKEI ADYLAAGKDE RARIRVEHII REDYLVEAME ILELYCDLLL ARFGLIQSM KELDSGLAES VSTLIWAAPR LQSEVAELKI VADQLCAKYS KEYGKLCRTN QIGTVNDRLM HKLSVEAPPK I LVERYLIE IAKNYNVPYE PDSVVMAEAP P

UniProtKB: IST1 homolog

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4
GridPretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 292 K / Instrument: FEI VITROBOT MARK I
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

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 3.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 26.85 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 4556
Startup modelType of model: INSILICO MODEL / In silico model: Smooth cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
FSC plot (resolution estimation)

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