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- EMDB-28722: CryoEM reconstruction of membrane-bound, right-handed CHMP1B+IST1... -

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Basic information

Entry
Database: EMDB / ID: EMD-28722
TitleCryoEM reconstruction of membrane-bound, right-handed CHMP1B+IST1 filament with brominated cholesterol probe
Map dataRight-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol.
Sample
  • Complex: Helical copolymer of CHMP1B and IST1 bound to a lipid membrane nanotube
    • Protein or peptide: CHMP1B
    • Protein or peptide: IST1
KeywordsESCRT / brominated / lipid / membrane / MEMBRANE 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 / endosome transport via multivesicular body sorting pathway ...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 / endosome transport via multivesicular body sorting pathway / cytoskeleton-dependent cytokinesis / regulation of centrosome duplication / collateral sprouting / 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 / multivesicular body membrane / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / regulation of mitotic spindle assembly / multivesicular body assembly / Flemming body / mitotic metaphase chromosome alignment / nucleus organization / viral budding via host ESCRT complex / positive regulation of proteolysis / endoplasmic reticulum-Golgi intermediate compartment / 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 / lysosomal membrane / cell division / 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: 4.3 Å
AuthorsMoss FR / Frost A
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117593 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM137109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082545 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM110772 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM089740 United States
Howard Hughes Medical Institute (HHMI) United States
Chan Zuckerberg Initiative 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
DepositionOct 29, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28722.png

Downloads & links

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Map

FileDownload / File: emd_28722.map.gz / Format: CCP4 / Size: 437.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRight-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 486 pix.
= 399.492 Å		0.82 Å/pix.
x 486 pix.
= 399.492 Å		0.82 Å/pix.
x 486 pix.
= 399.492 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.003
Minimum - Maximum-0.0069636484 - 0.0130933765
Average (Standard dev.)0.00017029585 (±0.0010411588)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions486486486
Spacing486486486
CellA=B=C: 399.492 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28722_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION post-processed, right-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated...

Fileemd_28722_additional_1.map
AnnotationRELION post-processed, right-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Right-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. No...

Fileemd_28722_additional_2.map
AnnotationRight-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. No helical symmetry has been applied.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Right-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. Half...

Fileemd_28722_half_map_1.map
AnnotationRight-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. Half 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Right-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. Half...

Fileemd_28722_half_map_2.map
AnnotationRight-handed, 18-subunit-per-turn, membrane-bound CHMP1B/IST1 with brominated cholesterol. Half 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Helical copolymer of CHMP1B and IST1 bound to a lipid membrane na...

EntireName: Helical copolymer of CHMP1B and IST1 bound to a lipid membrane nanotube
Components
  • Complex: Helical copolymer of CHMP1B and IST1 bound to a lipid membrane nanotube
    • Protein or peptide: CHMP1B
    • Protein or peptide: IST1

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Supramolecule #1: Helical copolymer of CHMP1B and IST1 bound to a lipid membrane na...

SupramoleculeName: Helical copolymer of CHMP1B and IST1 bound to a lipid membrane nanotube
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The lipid nanotube contains a brominated analog of Cholesterol
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: CHMP1B

MacromoleculeName: CHMP1B / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSNMEKHLFN LKFAAKELSR SAKKCDKEEK AEKAKIKKAI QKGNMEVARI HAENAIRQKN QAVNFLRMSA RVDAVAARVQ TAVTMGKVTK SMAGVVKSMD ATLKTMNLEK ISALMDKFEH QFETLDVQTQ QMEDTMSSTT TLTTPQNQVD MLLQEMADEA GLDLNMELPQ ...String:
MSNMEKHLFN LKFAAKELSR SAKKCDKEEK AEKAKIKKAI QKGNMEVARI HAENAIRQKN QAVNFLRMSA RVDAVAARVQ TAVTMGKVTK SMAGVVKSMD ATLKTMNLEK ISALMDKFEH QFETLDVQTQ QMEDTMSSTT TLTTPQNQVD MLLQEMADEA GLDLNMELPQ GQTGSVGTSV ASAEQDELSQ RLARLRDQV

UniProtKB: Charged multivesicular body protein 1b

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Macromolecule #2: IST1

MacromoleculeName: IST1 / type: protein_or_peptide / ID: 2 / Details: N-terminal domain / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLGSGFKAER LRVNLRLVIN RLKLLEKKKT ELAQKARKEI ADYLAAGKDE RARIRVEHII REDYLVEAME ILELYCDLLL ARFGLIQSMK ELDSGLAESV STLIWAAPRL QSEVAELKIV ADQLCAKYSK EYGKLCRTNQ IGTVNDRLMH KLSVEAPP

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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 292 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1855 / Average electron dose: 67.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / 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: 20.00 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 12868
Segment selectionNumber selected: 53787 / Software - Name: crYOLO
Startup modelType of model: INSILICO MODEL / In silico model: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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