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- EMDB-20327: Spastin Hexamer in complex with substrate peptide -

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Basic information

Entry
Database: EMDB / ID: EMD-20327
TitleSpastin Hexamer in complex with substrate peptide
Map data
Sample
  • Complex: Spastin Hexamer in complex with substrate peptide
    • Protein or peptide: Spastin
    • Protein or peptide: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
KeywordsAAA+ ATPase / Microtubule Severing / MOTOR PROTEIN
Function / homology
Function and homology information


cytokinetic process / microtubule-severing ATPase / microtubule severing ATPase activity / microtubule severing / positive regulation of microtubule depolymerization / endoplasmic reticulum tubular network / cytoskeleton-dependent cytokinesis / mitotic nuclear membrane reassembly / Sealing of the nuclear envelope (NE) by ESCRT-III / nuclear membrane reassembly ...cytokinetic process / microtubule-severing ATPase / microtubule severing ATPase activity / microtubule severing / positive regulation of microtubule depolymerization / endoplasmic reticulum tubular network / cytoskeleton-dependent cytokinesis / mitotic nuclear membrane reassembly / Sealing of the nuclear envelope (NE) by ESCRT-III / nuclear membrane reassembly / membrane fission / exit from mitosis / anterograde axonal transport / microtubule bundle formation / mitotic spindle disassembly / protein hexamerization / beta-tubulin binding / axonal transport of mitochondrion / positive regulation of cytokinesis / alpha-tubulin binding / mitotic cytokinesis / endoplasmic reticulum to Golgi vesicle-mediated transport / metabolic process / axon cytoplasm / isomerase activity / lipid droplet / axonogenesis / protein homooligomerization / spindle pole / midbody / cytoplasmic vesicle / microtubule binding / nuclear membrane / microtubule / endosome / axon / centrosome / protein-containing complex binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Spastin, chordate / Spastin / 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. ...Spastin, chordate / Spastin / 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
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHan H / Schubert HL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM112080 United States
CitationJournal: J Biol Chem / Year: 2020
Title: Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.
Authors: Han Han / Heidi L Schubert / John McCullough / Nicole Monroe / Michael D Purdy / Mark Yeager / Wesley I Sundquist / Christopher P Hill /
Abstract: Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic ...Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from , which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.
History
DepositionJun 20, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseDec 4, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0448
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0448
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pek
  • Surface level: 0.0448
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pen
  • Surface level: 0.0448
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20327.png

Downloads & links

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Map

FileDownload / File: emd_20327.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 0.0448 / Movie #1: 0.0448
Minimum - Maximum-0.08963044 - 0.17777756
Average (Standard dev.)0.00028883293 (±0.0042410973)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 270.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0561.0561.056
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z270.336270.336270.336
α/β/γ90.00090.00090.000
start NX/NY/NZ111-94150
NX/NY/NZ111123111
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0900.1780.000

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Supplemental data

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Sample components

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Entire : Spastin Hexamer in complex with substrate peptide

EntireName: Spastin Hexamer in complex with substrate peptide
Components
  • Complex: Spastin Hexamer in complex with substrate peptide
    • Protein or peptide: Spastin
    • Protein or peptide: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Spastin Hexamer in complex with substrate peptide

SupramoleculeName: Spastin Hexamer in complex with substrate peptide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Spastin

MacromoleculeName: Spastin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO / EC number: microtubule-severing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.498328 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAAKRSSGAA PAPASASAPA PVPGGEAERV RVFHKQAFEY ISIALRIDED EKAGQKEQAV EWYKKGIEEL EKGIAVIVTG QGEQCERAR RLQAKMMTNL VMAKDRLQLL ESGAVPKRKD PLTHTSNSLP RSKTVMKTGS AGLSGHHRAP SYSGLSMVSG V KQGSGPAP ...String:
MAAKRSSGAA PAPASASAPA PVPGGEAERV RVFHKQAFEY ISIALRIDED EKAGQKEQAV EWYKKGIEEL EKGIAVIVTG QGEQCERAR RLQAKMMTNL VMAKDRLQLL ESGAVPKRKD PLTHTSNSLP RSKTVMKTGS AGLSGHHRAP SYSGLSMVSG V KQGSGPAP TTHKGTPKTN RTNKPSTPTT ATRKKKDLKN FRNVDSNLAN LIMNEIVDNG TAVKFDDIAG QDLAKQALQE IV ILPSLRP ELFTGLRAPA RGLLLFGPPG NGKTMLAKAV AAESNATFFN ISAASLTSKY VGEGEKLVRA LFAVARELQP SII FIDEVD SLLCERREGE HDASRRLKTE FLIEFDGVQS AGDDRVLVMG ATNRPQELDE AVLRRFIKRV YVSLPNEETR LLLL KNLLC KQGSPLTQKE LAQLARMTDG YSGSDLTALA KDAALGPIRE LKPEQVKNMS ASEMRNIRLS DFTESLKKIK RSVSP QTLE AYIRWNKDFG DTTV

UniProtKB: Spastin

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Macromolecule #2: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU

MacromoleculeName: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.479453 KDa
SequenceString:
EYEYEYEYEY

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 119984

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