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- PDB-7zcg: CHMP2A-CHMP3 heterodimer (430 Angstrom diameter) -

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Basic information

Entry
Database: PDB / ID: 7zcg
TitleCHMP2A-CHMP3 heterodimer (430 Angstrom diameter)
Components
  • Charged multivesicular body protein 2a
  • Charged multivesicular body protein 3
KeywordsCYTOSOLIC PROTEIN / Cryo Electron Microscopy / Helical Reconstruction / Membrane-bound CHMP2A-CHMP3 filament / Negative-curvature membrane
Function / homology
Function and homology information


negative regulation of centriole elongation / regulation of endosome size / membrane invagination / : / amphisome membrane / multivesicular body-lysosome fusion / vesicle fusion with vacuole / suppression of viral release by host / ESCRT III complex disassembly / late endosome to lysosome transport ...negative regulation of centriole elongation / regulation of endosome size / membrane invagination / : / amphisome membrane / multivesicular body-lysosome fusion / vesicle fusion with vacuole / suppression of viral release by host / ESCRT III complex disassembly / late endosome to lysosome transport / ESCRT III complex / kinetochore microtubule / endosome transport via multivesicular body sorting pathway / regulation of centrosome duplication / nuclear membrane reassembly / Sealing of the nuclear envelope (NE) by ESCRT-III / midbody abscission / multivesicular body sorting pathway / membrane coat / membrane fission / plasma membrane repair / late endosome to vacuole transport / phosphatidylcholine binding / multivesicular body membrane / positive regulation of exosomal secretion / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body assembly / regulation of mitotic spindle assembly / Translation of Replicase and Assembly of the Replication Transcription Complex / exit from mitosis / Lysosome Vesicle Biogenesis / regulation of early endosome to late endosome transport / mitotic metaphase chromosome alignment / Macroautophagy / molecular function inhibitor activity / nucleus organization / ubiquitin-specific protease binding / viral budding via host ESCRT complex / positive regulation of cytokinesis / autophagosome membrane / autophagosome maturation / viral release from host cell / protein polymerization / Pyroptosis / nuclear pore / Endosomal Sorting Complex Required For Transport (ESCRT) / phosphatidylinositol-4,5-bisphosphate binding / multivesicular body / viral budding from plasma membrane / HCMV Late Events / macroautophagy / Late endosomal microautophagy / establishment of protein localization / Budding and maturation of HIV virion / protein homooligomerization / kinetochore / autophagy / protein transport / late endosome / nuclear envelope / Translation of Replicase and Assembly of the Replication Transcription Complex / midbody / cytoplasmic vesicle / early endosome / protein domain specific binding / lysosomal membrane / apoptotic process / extracellular exosome / identical protein binding / membrane / plasma membrane / cytosol
Similarity search - Function
Charged multivesicular body protein 2a / Charged multivesicular body protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAzad, K. / Desfosses, A. / Effantin, G. / Schoehn, G. / Weissenhorn, W.
Funding support France, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-14-CE09-0003-01 France
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0002-02 France
Agence Nationale de la Recherche (ANR)ANR-17-EURE-0003 France
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage.
Authors: Kimi Azad / Delphine Guilligay / Cecile Boscheron / Sourav Maity / Nicola De Franceschi / Guidenn Sulbaran / Gregory Effantin / Haiyan Wang / Jean-Philippe Kleman / Patricia Bassereau / Guy ...Authors: Kimi Azad / Delphine Guilligay / Cecile Boscheron / Sourav Maity / Nicola De Franceschi / Guidenn Sulbaran / Gregory Effantin / Haiyan Wang / Jean-Philippe Kleman / Patricia Bassereau / Guy Schoehn / Wouter H Roos / Ambroise Desfosses / Winfried Weissenhorn /
Abstract: The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, ...The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery.
History
DepositionMar 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Charged multivesicular body protein 2a
A: Charged multivesicular body protein 3


Theoretical massNumber of molelcules
Total (without water)35,7352
Polymers35,7352
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4090 Å2
ΔGint-44 kcal/mol
Surface area22350 Å2

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Components

#1: Protein Charged multivesicular body protein 2a / Chromatin-modifying protein 2a / CHMP2a / Putative breast adenocarcinoma marker BC-2 / Vacuolar ...Chromatin-modifying protein 2a / CHMP2a / Putative breast adenocarcinoma marker BC-2 / Vacuolar protein sorting-associated protein 2-1 / Vps2-1 / hVps2-1


Mass: 17305.488 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHMP2A, BC2, CHMP2 / Plasmid: pMAL-c5X / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: O43633
#2: Protein Charged multivesicular body protein 3 / Chromatin-modifying protein 3 / Neuroendocrine differentiation factor / Vacuolar protein sorting- ...Chromatin-modifying protein 3 / Neuroendocrine differentiation factor / Vacuolar protein sorting-associated protein 24 / hVps24


Mass: 18429.684 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHMP3, CGI149, NEDF, VPS24, CGI-149 / Plasmid: pProEX-HTb / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21Gold (DE3) / References: UniProt: Q9Y3E7

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Membrane-bound CHMP2A-CHMP3 filament (430 Angstrom Diameter) cryo-EM map
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
225 mMSodium ChlorideNaCl1
31 mMBeta-mercaptoethanolC2H6OS1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5 sec. / Electron dose: 24 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5028
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 25

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
11RELION3classification
12RELION33D reconstruction
13Cootmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 54.374 ° / Axial rise/subunit: 2.742 Å / Axial symmetry: C2
Particle selectionNum. of particles selected: 89122
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25353 / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6TZ4

6tz4

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