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- EMDB-20761: Katanin hexamer in the spiral conformation in complex with substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-20761
TitleKatanin hexamer in the spiral conformation in complex with substrate
Map dataKatanin hexamer in the spiral conformation in complex with substrate
Sample
  • Complex: Hexameric complex of C.elegans katanin bound to polyglutamate peptide
    • Protein or peptide: Meiotic spindle formation protein mei-1
    • Protein or peptide: Polyglutamate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordskatanin / microtubule-severing / MEI-1 / microtubule cytoskeleton / MOTOR PROTEIN
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 / meiotic spindle organization ...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 / meiotic spindle organization / female meiotic nuclear division / meiotic spindle / embryo development ending in birth or egg hatching / microtubule depolymerization / isomerase activity / spindle pole / spindle / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Meiotic spindle formation protein mei-1
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZehr EA / Roll-Mecak A
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseOct 9, 2019-
Map releaseOct 9, 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: 4.84
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.84
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6ugd
  • Surface level: 4.84
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20761.png

Downloads & links

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Map

FileDownload / File: emd_20761.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKatanin hexamer in the spiral conformation in complex with substrate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 224 pix.
= 241.92 Å		1.08 Å/pix.
x 224 pix.
= 241.92 Å		1.08 Å/pix.
x 224 pix.
= 241.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.84 / Movie #1: 4.84
Minimum - Maximum-16.68571 - 27.860405
Average (Standard dev.)0.00000000000413 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 241.92001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z241.920241.920241.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ224224224
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-16.68627.8600.000

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Supplemental data

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Sample components

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Entire : Hexameric complex of C.elegans katanin bound to polyglutamate peptide

EntireName: Hexameric complex of C.elegans katanin bound to polyglutamate peptide
Components
  • Complex: Hexameric complex of C.elegans katanin bound to polyglutamate peptide
    • Protein or peptide: Meiotic spindle formation protein mei-1
    • Protein or peptide: Polyglutamate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Hexameric complex of C.elegans katanin bound to polyglutamate peptide

SupramoleculeName: Hexameric complex of C.elegans katanin bound to polyglutamate peptide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 312 KDa

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Macromolecule #1: Meiotic spindle formation protein mei-1

MacromoleculeName: Meiotic spindle formation protein mei-1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: microtubule-severing ATPase
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 53.576336 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSFTMASMTG GQQMGRGSMN GDVQSVIRGY LERAQVAKTM SDAGRWNEAG DLLRQLMTDV KSCKISASNR DEHDARNTFL RALEANLKL VQQNVRDEDD LHEAMTRQSG SPEPPADPDV WSKPSPPLPS SSKFGATKKG VGAAGPRPRE ISKSTSSMST N PADVKPAN ...String:
GSFTMASMTG GQQMGRGSMN GDVQSVIRGY LERAQVAKTM SDAGRWNEAG DLLRQLMTDV KSCKISASNR DEHDARNTFL RALEANLKL VQQNVRDEDD LHEAMTRQSG SPEPPADPDV WSKPSPPLPS SSKFGATKKG VGAAGPRPRE ISKSTSSMST N PADVKPAN PTQGILPQNS AGDSFDASAY DAYIVQAVRG TMATNTENTM SLDDIIGMHD VKQVLHEAVT LPLLVPEFFQ GL RSPWKAM VLAGPPGTGK TLIARAIASE SSSTFFTVSS TDLSSKWRGD SEKIVRLLFE LARFYAPSII FIDQIDTLGG QRG NSGEHE ASRRVKSEFL VQMDGSQNKF DSRRVFVLAA TNIPWELDEA LRRRFEKRIF IPLPDIDARK KLIEKSMEGT PKSD EINYD DLAARTEGFS GADVVSLCRT AAINVLRRYD TKSLRGGELT AAMESLKAEL VRNIDFEAAL QAVSPSAGPD TMLKC KEWC DSFGAM

UniProtKB: Meiotic spindle formation protein mei-1

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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: synthetic construct (others)
Molecular weightTheoretical: 1.825609 KDa
SequenceString:
EEEEEEEEEE EEEE

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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: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
300.0 mMKClPotassium chloride
10.0 mMMgCl2Magnesium dichloride
2.0 mMC9H15O6PTCEP
1.0 mMC10H16N5O13P3ATP
GridModel: C-flat-2/1 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 279.15 K / Instrument: LEICA EM GP / Details: Load 5 ul sample, wait 10 sec, blot 4.5 sec.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 93.0 K / Max: 103.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 4 / Number real images: 5911 / Average exposure time: 10.0 sec. / Average electron dose: 73.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.8000000000000003 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFrames were aligned, dose weighted and summed using MotionCor2
Particle selectionNumber selected: 1876223 / Details: Particles were automatically picked
Startup modelType of model: EMDB MAP
EMDB ID:

8794


Details: Low-pass filtered to 35 A
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 40102
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 10 / Avg.num./class: 122136 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5wc0


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

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

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