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- PDB-6p07: Spastin hexamer in complex with substrate -

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Basic information

Entry
Database: PDB / ID: 6p07
TitleSpastin hexamer in complex with substrate
Components
  • Spastin
  • polyglutamate peptide
KeywordsMOTOR PROTEIN / AAA+ ATPase / Homohexamer / Microtubule Severing Enzyme
Function / homology
Function and homology information


microtubule-severing ATPase / microtubule severing ATPase activity / mitotic chromosome movement towards spindle pole / microtubule severing / mitotic spindle elongation / positive regulation of microtubule depolymerization / protein hexamerization / regulation of synapse structure or activity / lipid droplet / adult locomotory behavior ...microtubule-severing ATPase / microtubule severing ATPase activity / mitotic chromosome movement towards spindle pole / microtubule severing / mitotic spindle elongation / positive regulation of microtubule depolymerization / protein hexamerization / regulation of synapse structure or activity / lipid droplet / adult locomotory behavior / isomerase activity / spindle / chromosome / nervous system development / 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Spastin / Spastin
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSandate, C.R. / Szyk, A. / Zehr, E. / Roll-Mecak, A. / Lander, G.C.
Funding support United States, 2items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI
Revision 1.2Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Aug 14, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.5Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-20226
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spastin
B: Spastin
C: Spastin
D: Spastin
E: Spastin
F: Spastin
G: polyglutamate peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)330,67220
Polymers327,0567
Non-polymers3,61613
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Spastin


Mass: 54183.473 Da / Num. of mol.: 6 / Mutation: E583Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: spas, D-spastin, Dmel\CG5977, Dspastin, Spas, Spastin, CG5977, Dmel_CG5977
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A126GV13, UniProt: A0A0B4LHJ5*PLUS, microtubule-severing ATPase
#2: Protein/peptide polyglutamate peptide


Mass: 1954.722 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, 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: complex of spastin homohexamer bound to polyglutmate peptide
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 294 kDa/nm / Experimental value: YES
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPEs1
2300 mMpotassium chlorideKCl1
310 mMmagnesium chlorideMgCl21
45 mMDTT1
50.22 uMPolyglutamate peptide1
60.05 %LMNG detergent1
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were plasma-cleaned using a Solarus plasma cleaner (Gatan, Inc.)
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K
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
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 12 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2534
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 48 / Used frames/image: 1-48

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2Leginon3image acquisition
4CTFFINDCTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
Image processingDetails: 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).
CTF correctionDetails: CTF correction performed per-particle in CryoSparc / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2736865 / Details: Template-picked particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 488385 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00628384
ELECTRON MICROSCOPYf_angle_d0.79151491
ELECTRON MICROSCOPYf_dihedral_angle_d7.7511234
ELECTRON MICROSCOPYf_chiral_restr0.0462254
ELECTRON MICROSCOPYf_plane_restr0.0054215

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