[English] 日本語
Yorodumi
- PDB-6p07: Spastin hexamer in complex with substrate -

+
Open data


ID or keywords:

Loading...

-
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


positive regulation of microtubule depolymerization / microtubule-severing ATPase / 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 / microtubule-severing ATPase / 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
AAA+ ATPase domain / AAA-protein family signature. / AAA+ lid domain / ATPase family associated with various cellular activities (AAA) / AAA ATPase, AAA+ lid domain / MIT domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Spastin / MIT / ATPase, AAA-type, conserved site / ATPase, AAA-type, core
Spastin / Spastin
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.
Validation Report
SummaryFull reportAbout validation report
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

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-20226
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

#1: Protein/peptide
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 / Adenosine triphosphate / 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 / Magnesium
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
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

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPEs
2300 mMpotassium chlorideKCl
310 mMmagnesium chlorideMgCl2
45 mMDTT
50.22 uMPolyglutamate peptide
60.05 %LMNG detergent
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.

-
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 FIELDBright-field microscopy / 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 / 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

-
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

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00628384
f_angle_d0.79151491
f_dihedral_angle_d7.7511234
f_chiral_restr0.0462254
f_plane_restr0.0054215

+
About Yorodumi

-
News

-
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.: Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator

External links: EMDB at PDBe / Contact to PDBj

-
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.

External links: wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more