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- PDB-6uge: Katanin hexamer in the ring conformation in complex with substrate -

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Basic information

Entry
Database: PDB / ID: 6uge
TitleKatanin hexamer in the ring conformation in complex with substrate
Components
  • Meiotic spindle formation protein mei-1
  • Polyglutamate peptide
KeywordsMOTOR PROTEIN / katanin / microtubule-severing / MEI-1 / microtubule cytoskeleton
Function / homology
Function and homology information


negative regulation of meiotic spindle elongation / MATH domain binding / striated muscle myosin thick filament assembly / meiotic spindle disassembly / katanin complex / meiotic spindle pole / microtubule-severing ATPase / microtubule severing ATPase activity / microtubule severing / female meiotic nuclear division ...negative regulation of meiotic spindle elongation / MATH domain binding / striated muscle myosin thick filament assembly / meiotic spindle disassembly / katanin complex / meiotic spindle pole / microtubule-severing ATPase / microtubule severing ATPase activity / microtubule severing / female meiotic nuclear division / meiotic spindle organization / meiotic spindle / embryo development ending in birth or egg hatching / microtubule depolymerization / isomerase activity / spindle / spindle pole / microtubule cytoskeleton / microtubule binding / protein phosphatase binding / microtubule / molecular adaptor activity / cell division / centrosome / chromatin / ATP hydrolysis activity / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Katanin p60 subunit A1 / 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 / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain ...Katanin p60 subunit A1 / 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 / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Meiotic spindle formation protein mei-1
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZehr, E.A. / Roll-Mecak, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1 ZIA NS003165 United States
CitationJournal: Dev Cell / Year: 2020
Title: Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules.
Authors: Elena A Zehr / Agnieszka Szyk / Ewa Szczesna / Antonina Roll-Mecak /
Abstract: The AAA ATPase katanin severs microtubules. It is critical in cell division, centriole biogenesis, and neuronal morphogenesis. Its mutation causes microcephaly. The microtubule templates katanin ...The AAA ATPase katanin severs microtubules. It is critical in cell division, centriole biogenesis, and neuronal morphogenesis. Its mutation causes microcephaly. The microtubule templates katanin hexamerization and activates its ATPase. The structural basis for these activities and how they lead to severing is unknown. Here, we show that β-tubulin tails are necessary and sufficient for severing. Cryoelectron microscopy (cryo-EM) structures reveal the essential tubulin tail glutamates gripped by a double spiral of electropositive loops lining the katanin central pore. Each spiral couples allosterically to the ATPase and binds alternating, successive substrate residues, with consecutive residues coordinated by adjacent protomers. This tightly couples tail binding, hexamerization, and ATPase activation. Hexamer structures in different states suggest an ATPase-driven, ratchet-like translocation of the tubulin tail through the pore. A disordered region outside the AAA core anchors katanin to the microtubule while the AAA motor exerts the forces that extract tubulin dimers and sever the microtubule.
History
DepositionSep 26, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jan 15, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.4Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.value / _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_label_asym_id

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Meiotic spindle formation protein mei-1
B: Meiotic spindle formation protein mei-1
C: Meiotic spindle formation protein mei-1
D: Meiotic spindle formation protein mei-1
E: Meiotic spindle formation protein mei-1
F: Meiotic spindle formation protein mei-1
G: Polyglutamate peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)325,65916
Polymers323,0257
Non-polymers2,6339
Water0
1


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

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Components

#1: Protein
Meiotic spindle formation protein mei-1


Mass: 53576.336 Da / Num. of mol.: 6 / Mutation: E293Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mei-1, T01G9.5 / Plasmid: pDEST566 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P34808, microtubule-severing ATPase
#2: Protein/peptide Polyglutamate peptide


Mass: 1567.381 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Alamanda Polymers, CAS#26247-79-0 / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Hexameric complex of C.elegans katanin bound to polyglutamate peptide
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.312 MDa / Experimental value: NO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pDEST566
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2300 mMPotassium chlorideKCl1
310 mMMagnesium dichlorideMgCl21
42 mMTCEPC9H15O6P1
51 mMATPAdenosine triphosphateC10H16N5O13P31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 279.15 K / Details: Load 5 ul sample, wait 10 sec, blot 4.5 sec

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2800 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): 103 K / Temperature (min): 93 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 73 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 5911
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2Leginon3.4image acquisition
4Gctf1.06CTF correction
7Coot0.8.9model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.15.2model refinement
Image processingDetails: Frames were aligned, dose weighted and summed using MotionCor2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1876223 / Details: Particles were automatically picked
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108700 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5WC0

5wc0


Accession code: 5WC0 / Source name: PDB / Type: experimental model

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