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- PDB-5xmk: Cryo-EM structure of the ATP-bound Vps4 mutant-E233Q complex with... -

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Basic information

Entry
Database: PDB / ID: 5xmk
TitleCryo-EM structure of the ATP-bound Vps4 mutant-E233Q complex with Vta1 (masked)
Components
  • Vacuolar protein sorting-associated protein 4Vacuole
  • Vacuolar protein sorting-associated protein VTA1Vacuole
KeywordsPROTEIN TRANSPORT / ATPase / ESCRTIII / Vps4 / Vta1
Function / homology
Function and homology information


ESCRT IV complex / late endosome to lysosome transport via multivesicular body sorting pathway / intralumenal vesicle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / protein retention in Golgi apparatus / late endosome to vacuole transport via multivesicular body sorting pathway / sterol metabolic process / nuclear membrane reassembly / midbody abscission / vacuole organization ...ESCRT IV complex / late endosome to lysosome transport via multivesicular body sorting pathway / intralumenal vesicle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / protein retention in Golgi apparatus / late endosome to vacuole transport via multivesicular body sorting pathway / sterol metabolic process / nuclear membrane reassembly / midbody abscission / vacuole organization / multivesicular body sorting pathway / membrane fission / plasma membrane repair / late endosome to vacuole transport / multivesicular body assembly / reticulophagy / nucleus organization / lipid transport / endosomal transport / ATPase complex / ATPase activator activity / autophagosome maturation / nuclear pore / multivesicular body / macroautophagy / autophagy / protein-macromolecule adaptor activity / protein transport / midbody / endosome / endoplasmic reticulum / ATP hydrolysis activity / protein homodimerization activity / ATP binding / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Vta1/Callose synthase, N-terminal domain superfamily / Vta1/callose synthase, N-terminal / Vta1, C-terminal / Vacuolar protein sorting-associated protein Vta1-like / Vta1 like / Vta1 C-terminal domain / Vacuolar protein sorting-associated protein 4, MIT domain / MIT (microtubule interacting and transport) domain / MIT domain superfamily / Vps4 oligomerisation, C-terminal ...Vta1/Callose synthase, N-terminal domain superfamily / Vta1/callose synthase, N-terminal / Vta1, C-terminal / Vacuolar protein sorting-associated protein Vta1-like / Vta1 like / Vta1 C-terminal 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 / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Vacuolar protein sorting-associated protein 4 / Vacuolar protein sorting-associated protein VTA1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.18 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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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
G: Vacuolar protein sorting-associated protein VTA1
H: Vacuolar protein sorting-associated protein VTA1
I: Vacuolar protein sorting-associated protein VTA1
J: Vacuolar protein sorting-associated protein VTA1
K: Vacuolar protein sorting-associated protein VTA1
L: Vacuolar protein sorting-associated protein VTA1
M: Vacuolar protein sorting-associated protein VTA1
N: Vacuolar protein sorting-associated protein VTA1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)590,80619
Polymers588,27014
Non-polymers2,5365
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area42860 Å2
ΔGint-177 kcal/mol
Surface area104800 Å2

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Components

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


Mass: 48232.199 Da / Num. of mol.: 6
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: Protein
Vacuolar protein sorting-associated protein VTA1 / Vacuole / VPS20-associated protein 1


Mass: 37359.660 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: VTA1, YLR181C / Production host: Escherichia coli (E. coli) / References: UniProt: Q06263
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine 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 complexed with VTA1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
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 FIELDBright-field microscopy
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: 4.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106106 / Symmetry type: POINT
RefinementResolution: 4.18→166.32 Å / Cor.coef. Fo:Fc: 0.963 / SU B: 129.413 / SU ML: 1.435
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.35473 --
obs0.35473 60399 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 344.751 Å2
Baniso -1Baniso -2Baniso -3
1--5.01 Å20.31 Å23.57 Å2
2---7.75 Å22.5 Å2
3---12.76 Å2
Refinement stepCycle: 1 / Total: 18048
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0218332
ELECTRON MICROSCOPYr_bond_other_d0.0020.0217764
ELECTRON MICROSCOPYr_angle_refined_deg1.4531.99924804
ELECTRON MICROSCOPYr_angle_other_deg1.004341152
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.67952284
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.77825.259772
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.612153396
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.38115104
ELECTRON MICROSCOPYr_chiral_restr0.0910.22850
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02120258
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023688
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_it3.20534.819178
ELECTRON MICROSCOPYr_mcbond_other3.20534.8099177
ELECTRON MICROSCOPYr_mcangle_it5.99152.18511448
ELECTRON MICROSCOPYr_mcangle_other5.9952.18711449
ELECTRON MICROSCOPYr_scbond_it2.04234.8059154
ELECTRON MICROSCOPYr_scbond_other2.04234.8059154
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other4.13552.09613356
ELECTRON MICROSCOPYr_long_range_B_refined19.21566438
ELECTRON MICROSCOPYr_long_range_B_other19.21566439
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 4.181→4.483 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.539 4382 -
Rfree-0 -
obs--100 %

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