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- PDB-5xmi: Cryo-EM Structure of the ATP-bound VPS4 mutant-E233Q hexamer (masked) -

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Basic information

Entry
Database: PDB / ID: 5xmi
TitleCryo-EM Structure of the ATP-bound VPS4 mutant-E233Q hexamer (masked)
ComponentsVacuolar protein sorting-associated protein 4
KeywordsPROTEIN TRANSPORT / ATPase / EESCRTIII / Vps4
Function / homology
Function and homology information


ESCRT IV complex / Sealing of the nuclear envelope (NE) by ESCRT-III / late endosome to lysosome transport via multivesicular body sorting pathway / intralumenal vesicle formation / protein retention in Golgi apparatus / Endosomal Sorting Complex Required For Transport (ESCRT) / late endosome to vacuole transport via multivesicular body sorting pathway / sterol metabolic process / nuclear membrane reassembly / midbody abscission ...ESCRT IV complex / Sealing of the nuclear envelope (NE) by ESCRT-III / late endosome to lysosome transport via multivesicular body sorting pathway / intralumenal vesicle formation / protein retention in Golgi apparatus / Endosomal Sorting Complex Required For Transport (ESCRT) / late endosome to vacuole transport via multivesicular body sorting pathway / sterol metabolic process / nuclear membrane reassembly / midbody abscission / multivesicular body sorting pathway / vacuole organization / plasma membrane repair / membrane fission / late endosome to vacuole transport / multivesicular body assembly / reticulophagy / endosomal transport / nucleus organization / ATPase complex / autophagosome maturation / nuclear pore / macroautophagy / autophagy / protein transport / midbody / endosome / endoplasmic reticulum / protein homodimerization activity / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Vacuolar protein sorting-associated protein 4, MIT domain / MIT (microtubule interacting and transport) domain / MIT domain superfamily / Vps4 oligomerisation, C-terminal / MIT domain / Microtubule Interacting and Trafficking molecule domain / : / Vps4 C terminal oligomerisation domain / Helicase, Ruva Protein; domain 3 - #60 / AAA ATPase, AAA+ lid domain ...Vacuolar protein sorting-associated protein 4, MIT domain / MIT (microtubule interacting and transport) domain / MIT domain superfamily / Vps4 oligomerisation, C-terminal / MIT domain / Microtubule Interacting and Trafficking molecule domain / : / Vps4 C terminal oligomerisation domain / Helicase, Ruva Protein; domain 3 - #60 / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Vacuolar protein sorting-associated protein 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSun, S. / Li, L. / Yang, F. / Wang, X. / Fan, F. / Li, X. / Wang, H. / Sui, S.
CitationJournal: Nat Commun / Year: 2017
Title: Cryo-EM structures of the ATP-bound Vps4 hexamer and its complex with Vta1 at near-atomic resolution.
Authors: Shan Sun / Lin Li / Fan Yang / Xiaojing Wang / Fenghui Fan / Mengyi Yang / Chunlai Chen / Xueming Li / Hong-Wei Wang / Sen-Fang Sui /
Abstract: The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. ...The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4 hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4 subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.
History
DepositionMay 15, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 9, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2018Group: Data collection / Other / Refinement description / Category: cell / refine
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Vacuolar protein sorting-associated protein 4
B: Vacuolar protein sorting-associated protein 4
C: Vacuolar protein sorting-associated protein 4
D: Vacuolar protein sorting-associated protein 4
E: Vacuolar protein sorting-associated protein 4
F: Vacuolar protein sorting-associated protein 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,92911
Polymers289,3936
Non-polymers2,5365
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area25910 Å2
ΔGint-76 kcal/mol
Surface area92560 Å2

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Components

#1: Protein
Vacuolar protein sorting-associated protein 4 / DOA4-independent degradation protein 6 / Protein END13 / Vacuolar protein-targeting protein 10


Mass: 48232.199 Da / Num. of mol.: 6 / Mutation: E233Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Gene: VPS4, CSC1, DID6, END13, GRD13, VPL4, VPT10, YPR173C, P9705.10
Production host: Escherichia coli (E. coli) / References: UniProt: P52917
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vps4-E233Q hexamer / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: strain ATCC 204508 / S288c
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0145 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106918 / Symmetry type: POINT
RefinementResolution: 3.9→166.32 Å / Cor.coef. Fo:Fc: 0.929 / SU B: 109.676 / SU ML: 1.366
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.41184 --
obs0.41184 58178 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 232.884 Å2
Baniso -1Baniso -2Baniso -3
1--7.51 Å2-0.42 Å2-0.55 Å2
2---6.31 Å20.53 Å2
3---13.82 Å2
Refinement stepCycle: 1 / Total: 15000
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01915264
ELECTRON MICROSCOPYr_bond_other_d0.0010.0214628
ELECTRON MICROSCOPYr_angle_refined_deg1.1661.99520682
ELECTRON MICROSCOPYr_angle_other_deg0.891333876
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.37151908
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.4424.907648
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.231152748
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.9261596
ELECTRON MICROSCOPYr_chiral_restr0.0680.22346
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02116962
ELECTRON MICROSCOPYr_gen_planes_other0.0010.023144
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it1.3323.6747650
ELECTRON MICROSCOPYr_mcbond_other1.3323.6737649
ELECTRON MICROSCOPYr_mcangle_it2.55235.5089552
ELECTRON MICROSCOPYr_mcangle_other2.55235.5089553
ELECTRON MICROSCOPYr_scbond_it0.41123.5537614
ELECTRON MICROSCOPYr_scbond_other0.4123.5527615
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other1.10635.30511131
ELECTRON MICROSCOPYr_long_range_B_refined8.81628309
ELECTRON MICROSCOPYr_long_range_B_other8.81628310
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.9→4.33 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.559 4306 -
Rfree-0 -
obs--100 %

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