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- EMDB-20226: Spastin hexamer in complex with substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-20226
TitleSpastin hexamer in complex with substrate
Map data
Samplecomplex of spastin homohexamer bound to polyglutmate peptide:
Spastin / polyglutamate peptide / (ligand) x 3
Function / homology
Function and homology information


positive regulation of microtubule depolymerization / ec:5.6.1.1: / microtubule-severing ATPase activity / mitotic chromosome movement towards spindle pole / mitotic spindle elongation / regulation of synapse structure or activity / protein hexamerization / lipid droplet / adult locomotory behavior / isomerase activity ...positive regulation of microtubule depolymerization / ec:5.6.1.1: / microtubule-severing ATPase activity / mitotic chromosome movement towards spindle pole / mitotic spindle elongation / regulation of synapse structure or activity / protein hexamerization / lipid droplet / adult locomotory behavior / isomerase activity / spindle / nervous system development / chromosome / microtubule / microtubule binding / cell differentiation / centrosome / cell division / integral component of membrane / ATP binding / cytoplasm
Spastin / P-loop containing nucleoside triphosphate hydrolase / AAA-protein family signature. / MIT / AAA+ lid domain / ATPase, AAA-type, conserved site / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / AAA+ ATPase domain / MIT domain superfamily / AAA ATPase, AAA+ lid domain
Spastin
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSandate CR / Szyk A / Zehr E / Roll-Mecak A / Lander GC
CitationJournal: Nature Structural and Molecular Biology / Year: 2019
Title: An allosteric network in spastin couples multiple activities required for microtubule severing
Authors: Sandate CR / Szyk A / Zehr E / Lander GC / Roll-Mecak A
Validation ReportPDB-ID: 6p07

SummaryFull reportAbout validation report
DateDeposition: May 16, 2019 / Header (metadata) release: Jun 12, 2019 / Map release: Jun 12, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6p07
  • Surface level: 1.12
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20226.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.)
1.15 Å/pix.
x 256 pix.
= 294.4 Å
1.15 Å/pix.
x 256 pix.
= 294.4 Å
1.15 Å/pix.
x 256 pix.
= 294.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 1.12 / Movie #1: 1.12
Minimum - Maximum-2.6589453 - 5.7869997
Average (Standard dev.)0.0017488761 (±0.12507841)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.6595.7870.002

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Supplemental data

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Sample components

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Entire complex of spastin homohexamer bound to polyglutmate peptide

EntireName: complex of spastin homohexamer bound to polyglutmate peptide
Number of components: 6

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Component #1: protein, complex of spastin homohexamer bound to polyglutmate peptide

ProteinName: complex of spastin homohexamer bound to polyglutmate peptide
Recombinant expression: No
MassExperimental: 294 MDa
SourceSpecies: Drosophila melanogaster (fruit fly)

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Component #2: protein, Spastin

ProteinName: Spastin / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 54.183473 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, polyglutamate peptide

ProteinName: polyglutamate peptide / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 1.954722 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)

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

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.2 mg/mL / pH: 7.5
Support filmGrids were plasma-cleaned using a Solarus plasma cleaner (Gatan, Inc.)
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 90 %
Details: Sample was blotted approximately 4 seconds using Whatman No. 1 filter paper before plunge-freezing..

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 52 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (70.0 - 70.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2534 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 488385
Details: Collected in counting mode, 48 frames movie-1, exposure time 12 s (250 ms frames), exposure rate of ~5.6 e- pixel-1 s-1, total exposure of ~52 e- angstrom-2 (1.08 e- angstrom-2 frame-1).
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION
CTF correction: CTF correction performed per-particle in CryoSparc
Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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