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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 dataCryo-EM map of spastin hexamer bound to substrate peptide.
Sample
  • Complex: complex of spastin homohexamer bound to polyglutmate peptide
    • Protein or peptide: Spastin
    • Protein or peptide: polyglutamate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ ATPase / Homohexamer / Microtubule Severing Enzyme / MOTOR PROTEIN
Function / homology
Function and homology information


microtubule-severing ATPase / microtubule severing ATPase activity / mitotic chromosome movement towards spindle pole / microtubule severing / positive regulation of microtubule depolymerization / mitotic spindle elongation / protein hexamerization / regulation of synapse structure or activity / isomerase activity / lipid droplet ...microtubule-severing ATPase / microtubule severing ATPase activity / mitotic chromosome movement towards spindle pole / microtubule severing / positive regulation of microtubule depolymerization / mitotic spindle elongation / protein hexamerization / regulation of synapse structure or activity / isomerase activity / lipid droplet / adult locomotory behavior / spindle / nervous system development / chromosome / microtubule binding / microtubule / cell differentiation / cell division / centrosome / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytoplasm
Similarity search - Function
Spastin / 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 ...Spastin / 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
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSandate CR / Szyk A
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP2EB020402 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: An allosteric network in spastin couples multiple activities required for microtubule severing.
Authors: Colby R Sandate / Agnieszka Szyk / Elena A Zehr / Gabriel C Lander / Antonina Roll-Mecak /
Abstract: The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal ...The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.
History
DepositionMay 16, 2019-
Header (metadata) releaseJun 12, 2019-
Map releaseJun 12, 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: 1.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.12
  • Imaged by UCSF Chimera
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  • 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

emd_20226.png

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)
AnnotationCryo-EM map of spastin hexamer bound to substrate peptide.
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

Histogram

Histogram (log scale)
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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Additional map: Unsharpened map

Fileemd_20226_additional.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_20226_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_20226_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: complex of spastin homohexamer bound to polyglutmate peptide
    • Protein or peptide: Spastin
    • Protein or peptide: polyglutamate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: complex of spastin homohexamer bound to polyglutmate peptide

SupramoleculeName: complex of spastin homohexamer bound to polyglutmate peptide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 294 kDa/nm

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Macromolecule #1: Spastin

MacromoleculeName: Spastin / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: microtubule-severing ATPase
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 54.183473 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPQGSKAANR PGGGYSPGPG DPLLAKQKHH HRRAFEYISK ALKIDEENEG HKELAIELYR KGIKELEDGI AVDCWSGRGD VWDRAQRLH DKMQTNLSMA RDRLHFLASG RKLTIGSKRP VNLAVANKSQ TLPRNLGSKT SVGAVQRQPV KTAATPPAVR R QFSSGRNT ...String:
GPQGSKAANR PGGGYSPGPG DPLLAKQKHH HRRAFEYISK ALKIDEENEG HKELAIELYR KGIKELEDGI AVDCWSGRGD VWDRAQRLH DKMQTNLSMA RDRLHFLASG RKLTIGSKRP VNLAVANKSQ TLPRNLGSKT SVGAVQRQPV KTAATPPAVR R QFSSGRNT PPQRSRTPIN NNGPSGSGAS TPVVSVKGVE QKLVQLILDE IVEGGAKVEW TDIAGQDVAK QALQEMVILP SV RPELFTG LRAPAKGLLL FGPPGNGKTL LARAVATECS ATFLNISAAS LTSKYVGDGE KLVRALFAVA RHMQPSIIFI DQV DSLLSE RSSSEHEASR RLKTEFLVEF DGLPGNPDGD RIVVLAATNR PQELDEAALR RFTKRVYVSL PDEQTRELLL NRLL QKQGS PLDTEALRRL AKITDGYSGS DLTALAKDAA LEPIRELNVE QVKCLDISAM RAITEQDFHS SLKRIRRSVA PQSLN SYEK WSQDYGDITI

UniProtKB: Spastin

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Macromolecule #2: polyglutamate peptide

MacromoleculeName: polyglutamate peptide / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 1.954722 KDa
SequenceString:
EEEEEEEEEE EEEEE

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPEs
300.0 mMpotassium chlorideKCl
10.0 mMmagnesium chlorideMgCl2
5.0 mMDTT
0.22 uMPolyglutamate peptide
0.05 %LMNG detergent
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
Details: Grids were plasma-cleaned using a Solarus plasma cleaner (Gatan, Inc.)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Sample was blotted approximately 4 seconds using Whatman No. 1 filter paper before plunge-freezing..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-48 / Number grids imaged: 2 / Number real images: 2534 / Average exposure time: 12.0 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsCollected 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).
Particle selectionNumber selected: 2736865 / Details: Template-picked particles
Startup modelType of model: OTHER
Details: An ab initio model generated from ~1.26 million template-picked particles cryoSPARC was used as a startup model.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 488385
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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