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- PDB-6ugd: Katanin hexamer in the spiral conformation in complex with substrate -

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Basic information

Entry
Database: PDB / ID: 6ugd
TitleKatanin hexamer in the spiral 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 / katanin complex / meiotic spindle disassembly / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / female meiotic nuclear division / meiotic spindle organization / striated muscle myosin thick filament assembly ...negative regulation of meiotic spindle elongation / MATH domain binding / katanin complex / meiotic spindle disassembly / microtubule severing / microtubule-severing ATPase / microtubule-severing ATPase activity / female meiotic nuclear division / meiotic spindle organization / striated muscle myosin thick filament assembly / embryo development ending in birth or egg hatching / microtubule depolymerization / protein hexamerization / cytoplasmic microtubule organization / isomerase activity / spindle / spindle pole / chromosome / microtubule / microtubule binding / protein phosphatase binding / ATPase activity / centrosome / cell division / ATP binding / identical protein binding / nucleus
ATPase, AAA-type, core / AAA+ ATPase domain / AAA-protein family signature. / AAA+ lid domain / ATPase family associated with various cellular activities (AAA) / AAA ATPase, AAA+ lid domain / Katanin p60 subunit A1 / P-loop containing nucleoside triphosphate hydrolase / ATPase, AAA-type, conserved site
Meiotic spindle formation protein mei-1
Biological speciesCaenorhabditis elegans (roundworm)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZehr, E.A. / Roll-Mecak, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke1 ZIA NS003165 United States
CitationJournal: To Be Published
Title: Katanin grips the b-tubulin tail through an electropositive double spiral to sever microtubules
Authors: Zehr, E.A. / Szyk, A. / Szczesna, E. / Roll-Mecak, A.
Validation Report
SummaryFull reportAbout validation report
History
DepositionSep 26, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release

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Structure visualization

Movie
  • 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)326,44818
Polymers323,2847
Non-polymers3,16511
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
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 (roundworm) / 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: 1825.609 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / Details: Alamanda Polymers, CAS#26247-79-0
#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.: 5 / Source method: obtained synthetically / Formula: Mg / Magnesium
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 (roundworm)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pDEST566
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPESC8H18N2O4S
2300 mMPotassium chlorideKCl
310 mMMagnesium dichlorideMgCl2
42 mMTCEPC9H15O6P
51 mMATPAdenosine triphosphateC10H16N5O13P3
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 / 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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40102 / 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

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