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- EMDB-6461: Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer -

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Basic information

Entry
Database: EMDB / ID: EMD-6461
TitleElectron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer
Map dataReconstruction of IST1NTD-CHMP1B copolymer
Sample
  • Sample: ESCRT-III copolymer of IST1 and CHMP1B
  • Protein or peptide: IST1
  • Protein or peptide: Charged multivesicular body protein 1b
KeywordsESCRT-III / IST1 / CHMP1B / membrane tubulation / helical filament
Function / homology
Function and homology information


viral capsid secondary envelopment / MIT domain binding / abscission / amphisome membrane / multivesicular body-lysosome fusion / vesicle fusion with vacuole / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule ...viral capsid secondary envelopment / MIT domain binding / abscission / amphisome membrane / multivesicular body-lysosome fusion / 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 / Sealing of the nuclear envelope (NE) by ESCRT-III / nuclear membrane reassembly / positive regulation of collateral sprouting / midbody abscission / multivesicular body sorting pathway / membrane coat / membrane fission / plasma membrane repair / late endosome to vacuole transport / multivesicular body membrane / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body assembly / regulation of mitotic spindle assembly / Flemming body / nucleus organization / mitotic metaphase chromosome alignment / viral budding via host ESCRT complex / autophagosome maturation / autophagosome membrane / positive regulation of proteolysis / viral release from host cell / endoplasmic reticulum-Golgi intermediate compartment / nuclear pore / multivesicular body / viral budding from plasma membrane / establishment of protein localization / protein localization / kinetochore / autophagy / azurophil granule lumen / protein transport / nuclear envelope / midbody / endosome membrane / cadherin binding / cell division / lysosomal membrane / protein domain specific binding / intracellular membrane-bounded organelle / centrosome / chromatin / Neutrophil degranulation / protein-containing complex binding / extracellular exosome / extracellular region / nucleoplasm / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Vacuolar protein sorting-associated protein Ist1 / Regulator of Vps4 activity in the MVB pathway / Vacuolar protein sorting-associated protein IST1-like / 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: 4.0 Å
AuthorsMcCullough J / Clippinger AK / Talledge N / Skowyra ML / Saunders MG / Naismith TV / Colf LA / Afonine P / Arthur C / Sundquist WI ...McCullough J / Clippinger AK / Talledge N / Skowyra ML / Saunders MG / Naismith TV / Colf LA / Afonine P / Arthur C / Sundquist WI / Hanson PI / Frost A
CitationJournal: Science / Year: 2015
Title: Structure and membrane remodeling activity of ESCRT-III helical polymers.
Authors: John McCullough / Amy K Clippinger / Nathaniel Talledge / Michael L Skowyra / Marissa G Saunders / Teresa V Naismith / Leremy A Colf / Pavel Afonine / Christopher Arthur / Wesley I Sundquist ...Authors: John McCullough / Amy K Clippinger / Nathaniel Talledge / Michael L Skowyra / Marissa G Saunders / Teresa V Naismith / Leremy A Colf / Pavel Afonine / Christopher Arthur / Wesley I Sundquist / Phyllis I Hanson / Adam Frost /
Abstract: The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal ...The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises "open" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of "closed" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.
History
DepositionSep 9, 2015-
Header (metadata) releaseOct 14, 2015-
Map releaseDec 16, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jc1
  • Surface level: 12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jc1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6461.png

Downloads & links

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Map

FileDownload / File: emd_6461.map.gz / Format: CCP4 / Size: 126.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of IST1NTD-CHMP1B copolymer
Voxel sizeX=Y=Z: 1.2 Å
Density
Contour LevelBy AUTHOR: 12.0 / Movie #1: 12
Minimum - Maximum-40.7751503 - 46.757438659999998
Average (Standard dev.)0.01477195 (±3.64489412)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin151910
Dimensions324324324
Spacing324324324
CellA=B=C: 388.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z388.800388.800388.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS191510
NC/NR/NS324324324
D min/max/mean-40.77546.7570.015

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Supplemental data

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Sample components

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Entire : ESCRT-III copolymer of IST1 and CHMP1B

EntireName: ESCRT-III copolymer of IST1 and CHMP1B
Components
  • Sample: ESCRT-III copolymer of IST1 and CHMP1B
  • Protein or peptide: IST1
  • Protein or peptide: Charged multivesicular body protein 1b

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Supramolecule #1000: ESCRT-III copolymer of IST1 and CHMP1B

SupramoleculeName: ESCRT-III copolymer of IST1 and CHMP1B / type: sample / ID: 1000
Oligomeric state: 2-stranded helical filament composed of one strand of IST1 subunits and one strand of CHMP1B subunits
Number unique components: 2

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Macromolecule #1: IST1

MacromoleculeName: IST1 / type: protein_or_peptide / ID: 1
Name.synonym: Increased Sodium Tolerance 1, hIST1, Putatuve MAPK-activating protein PM28
Details: IST1 N-terminal domain, residues 1-189 / Oligomeric state: Polymer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 22 KDa / Theoretical: 22 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: BL21 (RIPL) / Recombinant plasmid: pGEX-2T-TEV
SequenceUniProtKB: IST1 homolog

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Macromolecule #2: Charged multivesicular body protein 1b

MacromoleculeName: Charged multivesicular body protein 1b / type: protein_or_peptide / ID: 2
Name.synonym: CHMP1B, Chromatin-modifying protein 1b (CHMP1.5), Vacuolar protein sorting-associated protein 46-2 (hVps46-2)
Details: full-length CHMP1B / Oligomeric state: Polymer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 22 KDa / Theoretical: 22 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: BL21 (RIPL) / Recombinant plasmid: pGEX-2T-TEV
SequenceUniProtKB: Charged multivesicular body protein 1b

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 8 / Details: 25 mM Tris, 25 mM NaCl
GridDetails: 3.5 uL of pelleted and resuspended liposome-nucleated IST1NTD-CHMP1B copolymers were applied to glow-discharged Quantifoil holey carbon grids (2 micron hole size, 2-4 micron spacing, 200 mesh).
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I
Method: Blotted 7-9 seconds (-2 mm offset) and plunge-frozen

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Electron microscopy #1

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Microscopy ID1
DateJun 1, 2013
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 2493 / Average electron dose: 10 e/Å2
Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs ...Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs collected on a JEM3200FSC microscope with a DE-12 direct electron detector.
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Electron microscopy #2

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID2
DateJul 1, 2013
Image recordingCategory: FILM / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 2493 / Average electron dose: 15 e/Å2
Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs ...Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs collected on a JEM3200FSC microscope with a DE-12 direct electron detector.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #3

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.40 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
Microscopy ID3
DateAug 1, 2013
Image recordingCategory: FILM / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 2493 / Average electron dose: 20 e/Å2
Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs ...Details: 118,467 particles were selected from 2454 micrographs collected on a Titan Krios microscope with a Falcon I direct electron detector; 12,142 particles were selected from 39 micrographs collected on a JEM3200FSC microscope with a DE-12 direct electron detector.
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each particle as implemented in RELION
Final reconstructionApplied symmetry - Helical parameters - Δz: 2.96 Å
Applied symmetry - Helical parameters - Δ&Phi: 21.060 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2, IHRSR, SPIDER, RELION
Details: 51 copies (3 complete turns) of the asymmetric RELION reconstruction were transformed according to the helical symmetry, resampled on the original grid, and summed together, with Iterative ...Details: 51 copies (3 complete turns) of the asymmetric RELION reconstruction were transformed according to the helical symmetry, resampled on the original grid, and summed together, with Iterative Helical Real Space Reconstruction (IHRSR) single-particle algorithm as implemented in SPIDER and high-resolution refinement in RELION.
DetailsParticles were picked manually using the e2helixboxer.py function of EMAN2. Particles were aligned with IHRSR initially, followed by high resolution asymmetric refinement in RELION, followed by helical averaging in real space.

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Atomic model buiding 1

Initial modelPDB ID:

3frr


Chain - Chain ID: A
SoftwareName: Coot, Chimera, NAMD MDFF, Rosetta
Details3FRR was docked manually into the segmented density using Chimera. Regions with poor fit to density were identified using the Rosetta loops-from-density algorithm and iteratively fitted using alternating cycles of Rosetta's rebuild and refine protocol and manual refinement in Coot. The full ring of IST1 structures was then refined using Rosetta's symmetry constraints. Finally, backbone hydrogen bonds in the helical regions were constrained and two cycles of loop rebuilding with constraints were performed. The IST1 model was then combined with the CHMP1B model to form a heterodimer and this structure was refined by iterating between manual rebuilding and refinement using MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Molprobity validation, cross-correlation
Output model

PDB-3jc1:
Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer

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