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- EMDB-20588: CryoEM reconstruction of membrane-bound ESCRT-III filament compos... -

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Basic information

Entry
Database: EMDB / ID: EMD-20588
TitleCryoEM reconstruction of membrane-bound ESCRT-III filament composed of CHMP1B+IST1 (right-handed)
Map dataRELION postprocessed map with helical symmetry applied to the entire map
Sample
  • Complex: membrane-bound ESCRT-III copolymer filament composed of CHMP1B and IST1
    • Protein or peptide: Charged multivesicular body protein 1b
    • 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 / multivesicular body-lysosome fusion / amphisome membrane / vesicle fusion with vacuole / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule / cytoskeleton-dependent cytokinesis ...MIT domain binding / abscission / multivesicular body-lysosome fusion / amphisome membrane / vesicle fusion with vacuole / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule / cytoskeleton-dependent cytokinesis / endosome transport via multivesicular body sorting pathway / collateral sprouting / regulation of centrosome duplication / nuclear membrane reassembly / positive regulation of collateral sprouting / Sealing of the nuclear envelope (NE) by ESCRT-III / midbody abscission / multivesicular body sorting pathway / plasma membrane repair / membrane coat / membrane fission / late endosome to vacuole transport / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body membrane / multivesicular body assembly / regulation of mitotic spindle assembly / Flemming body / mitotic metaphase chromosome alignment / nucleus organization / viral budding via host ESCRT complex / endoplasmic reticulum-Golgi intermediate compartment / positive regulation of proteolysis / autophagosome membrane / autophagosome maturation / nuclear pore / multivesicular body / viral budding from plasma membrane / establishment of protein localization / kinetochore / autophagy / azurophil granule lumen / protein localization / protein transport / nuclear envelope / midbody / endosome membrane / cadherin binding / protein domain specific binding / cell division / lysosomal membrane / intracellular membrane-bounded organelle / centrosome / Neutrophil degranulation / protein-containing complex binding / chromatin / extracellular exosome / extracellular region / nucleoplasm / identical protein binding / plasma membrane / 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 / Snf7 family / Snf7
Similarity search - Domain/homology
IST1 homolog / Charged multivesicular body protein 1b
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 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 10, 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: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tz4
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tz4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20588.png

Downloads & links

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Map

FileDownload / File: emd_20588.map.gz / Format: CCP4 / Size: 166.4 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.)
1.22 Å/pix.
x 352 pix.
= 429.44 Å		1.22 Å/pix.
x 352 pix.
= 429.44 Å		1.22 Å/pix.
x 352 pix.
= 429.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.22 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.020064756 - 0.047598813
Average (Standard dev.)0.0004473235 (±0.0036865792)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 429.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z429.440429.440429.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0200.0480.000

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Supplemental data

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Mask #1

Fileemd_20588_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Additional map: RELION Refine3D filtered map with no symmetry applied (C1 map)

Fileemd_20588_additional_1.map
AnnotationRELION Refine3D filtered map with no symmetry applied (C1 map)
Projections & Slices
AxesZYX

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Histogram

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

Fileemd_20588_additional_2.map
AnnotationRELION Refine3D filtered map with helical symmetry imposed on the central 40%
Projections & Slices
AxesZYX

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Additional map: RELION Refine3D unfiltered half map1 with no symmetry...

Fileemd_20588_additional_3.map
AnnotationRELION Refine3D unfiltered half map1 with no symmetry applied (C1 half1 map)
Projections & Slices
AxesZYX

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Histogram

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Additional map: RELION Refine3D unfiltered half map2 with no symmetry...

Fileemd_20588_additional_4.map
AnnotationRELION Refine3D unfiltered half map2 with no symmetry applied (C1 half2 map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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

Fileemd_20588_half_map_1.map
AnnotationRELION Refine3D unfiltered half map1 with helical symmetry imposed on the central 40%
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_20588_half_map_2.map
AnnotationRELION Refine3D unfiltered half map2 with helical symmetry imposed on the central 40%
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : membrane-bound ESCRT-III copolymer filament composed of CHMP1B an...

EntireName: membrane-bound ESCRT-III copolymer filament composed of CHMP1B and IST1
Components
  • Complex: membrane-bound ESCRT-III copolymer filament composed of CHMP1B and IST1
    • Protein or peptide: Charged multivesicular body protein 1b
    • Protein or peptide: IST1 homolog

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Supramolecule #1: membrane-bound ESCRT-III copolymer filament composed of CHMP1B an...

SupramoleculeName: membrane-bound ESCRT-III copolymer filament composed of CHMP1B and IST1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Charged multivesicular body protein 1b

MacromoleculeName: Charged multivesicular body protein 1b / type: protein_or_peptide / ID: 1 / Number of copies: 36 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.140354 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSNMEKHLFN LKFAAKELSR SAKKCDKEEK AEKAKIEKAI QKGNMEVARI HAENAIRQKN QAVNFLRMSA RVDAVAARVQ TAVTMGKVT KSMAGVVKSM DATLKTMNLE KISALMDKFE HQFETLDVQT QQMEDTMSST TTLTTPQNQV DMLLQEMADE A GLDLNMEL ...String:
MSNMEKHLFN LKFAAKELSR SAKKCDKEEK AEKAKIEKAI QKGNMEVARI HAENAIRQKN QAVNFLRMSA RVDAVAARVQ TAVTMGKVT KSMAGVVKSM DATLKTMNLE KISALMDKFE HQFETLDVQT QQMEDTMSST TTLTTPQNQV DMLLQEMADE A GLDLNMEL PQGQTGSVGT SVASAEQDEL SQRLARLRDQ V

UniProtKB: Charged multivesicular body protein 1b

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Macromolecule #2: IST1 homolog

MacromoleculeName: IST1 homolog / type: protein_or_peptide / ID: 2 / Number of copies: 36 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.574281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLGSGFKAER LRVNLRLVIN RLKLLEKKKT 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 III
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 POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 2.96 Å
Applied symmetry - Helical parameters - Δ&Phi: 20.02 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 61974
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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