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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
SampleSpastin Hexamer in complex with substrate peptide
  • Spastin
  • substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU
  • (ligand) x 3
Function / homology
Function and homology information


membrane fission / cytokinetic process / nuclear envelope reassembly / positive regulation of microtubule depolymerization / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / cytoskeleton-dependent cytokinesis / endoplasmic reticulum tubular network / mitotic spindle disassembly ...membrane fission / cytokinetic process / nuclear envelope reassembly / positive regulation of microtubule depolymerization / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / cytoskeleton-dependent cytokinesis / endoplasmic reticulum tubular network / mitotic spindle disassembly / exit from mitosis / axonal transport of mitochondrion / anterograde axonal transport / microtubule bundle formation / positive regulation of cytokinesis / protein hexamerization / beta-tubulin binding / alpha-tubulin binding / cytoplasmic microtubule organization / metabolic process / lipid droplet / axon cytoplasm / mitotic cytokinesis / isomerase activity / axonogenesis / spindle / midbody / endoplasmic reticulum to Golgi vesicle-mediated transport / protein homooligomerization / cytoplasmic vesicle / microtubule / microtubule binding / nuclear membrane / endosome / ATPase activity / protein-containing complex binding / centrosome / endoplasmic reticulum membrane / perinuclear region of cytoplasm / extracellular exosome / integral component of membrane / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
AAA ATPase, AAA+ lid domain / Spastin, chordate / P-loop containing nucleoside triphosphate hydrolase / Spastin / Vps4 oligomerisation, C-terminal / MIT / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain
Spastin
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHan H / Schubert HL / McCullough J / Monroe N / Sundquist WI / Hill CP
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM112080 United States
CitationJournal: J. Biol. Chem. / Year: 2019
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 a1A/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, while 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 Drosophila melanogaster, 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.
Validation ReportPDB-ID: 6pek

SummaryFull report
PDB-ID: 6pen

SummaryFull report
About validation report
History
DepositionJun 20, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseDec 4, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing 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
  • Download
  • 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
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6pen
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6pek
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 270.336 Å
1.06 Å/pix.
x 256 pix.
= 270.336 Å
1.06 Å/pix.
x 256 pix.
= 270.336 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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 / Number of components: 6

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Component #1: protein, Spastin Hexamer in complex with substrate peptide

ProteinName: Spastin Hexamer in complex with substrate peptide / Recombinant expression: No
SourceSpecies: Homo sapiens (human)

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Component #2: protein, Spastin

ProteinName: Spastin / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 54.498328 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU

ProteinName: substrate peptide, TYR-GLU-TYR-GLU-TYR-GLU-TYR-GLU / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 1.479453 kDa
SourceSpecies: synthetic construct (others)

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Component #4: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #5: ligand, BERYLLIUM TRIFLUORIDE ION

LigandName: BERYLLIUM TRIFLUORIDE ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 6.600705 MDa

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Component #6: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 57 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 119984
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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