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- EMDB-10625: Cin8 motor domain (short loop 8) bound to microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-10625
TitleCin8 motor domain (short loop 8) bound to microtubules
Map dataCin8 motor domain (short loop 8) bound to microtubules
Sample
  • Complex: ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN
    • Protein or peptide: Cin8, microtubules
KeywordsKinesin / Cin8 / Motor domain / Microtubules / MOTOR PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsSiegler N / Singh SK / Zalk R / Debs GE / Sindelar CV / Gheber L / Zarivach R
Funding support Israel, 1 items
OrganizationGrant numberCountry
Israel Science Foundation386/18 Israel
CitationJournal: Sci Adv / Year: 2024
Title: Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin.
Authors: Sudhir K Singh / Nurit Siegler / Himanshu Pandey / Neta Yanir / Mary Popov / Alina Goldstein-Levitin / Mayan Sadan / Garrett Debs / Raz Zarivach / Gabriel A Frank / Itamar Kass / Charles V ...Authors: Sudhir K Singh / Nurit Siegler / Himanshu Pandey / Neta Yanir / Mary Popov / Alina Goldstein-Levitin / Mayan Sadan / Garrett Debs / Raz Zarivach / Gabriel A Frank / Itamar Kass / Charles V Sindelar / Ran Zalk / Larisa Gheber /
Abstract: Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent ...Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of β-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.
History
DepositionJan 18, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10625.png

Downloads & links

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Map

FileDownload / File: emd_10625.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCin8 motor domain (short loop 8) bound to microtubules
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 248 pix.
= 272.8 Å		1.1 Å/pix.
x 248 pix.
= 272.8 Å		1.1 Å/pix.
x 248 pix.
= 272.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.081666864 - 0.29207584
Average (Standard dev.)-0.008126179 (±0.013407394)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions248248248
Spacing248248248
CellA=B=C: 272.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z248248248
origin x/y/z0.0000.0000.000
length x/y/z272.800272.800272.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS248248248
D min/max/mean-0.0820.292-0.008

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Supplemental data

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Sample components

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Entire : ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN

EntireName: ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN
Components
  • Complex: ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN
    • Protein or peptide: Cin8, microtubules

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Supramolecule #1: ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN

SupramoleculeName: ALPHA and Beta TUBULIN WITH CIN8 MOTOR DOMAIN / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: ADDED 0.1 mM AMP-PNP, 10 micromolar Taxol
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Cin8, microtubules

MacromoleculeName: Cin8, microtubules / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPAENQNTGQ DRSSNSISKN GNSQVGCHTV PNEELNITVA VRCRGRNERE ISMKSSVVVN VPDITGSKEI SINTTGDTGI TAQMNAKRYT VDKVFGPGAS QDLIFDEVAG PLFQDFIKGY NCTVLVYGMT STGKTYTMTG DEKLYNGELS DAAGIIPRVL LKLFDTLELQ ...String:
MPAENQNTGQ DRSSNSISKN GNSQVGCHTV PNEELNITVA VRCRGRNERE ISMKSSVVVN VPDITGSKEI SINTTGDTGI TAQMNAKRYT VDKVFGPGAS QDLIFDEVAG PLFQDFIKGY NCTVLVYGMT STGKTYTMTG DEKLYNGELS DAAGIIPRVL LKLFDTLELQ QNDYVVKCSF IELYNEELKD LLDSNSNGSS NTGFDGQFMK KLRIFDNNNN NSSIYIQNLQ EFHITNAMEG LNLLQKGLKH RQVASTKMND FSSRSHTIFT ITLYKKHQDE LFRISKMNLV DLAGSENINR SGALNQRAKE AGSINQSLLT LGRVINALVD KSGHIPFRES KLTRLLQDSL GGNTKTALIA TISPAKVTSE ETCSTLEYAS KAKNIKNKPQ LGSFIMKDIL LEHHHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.9
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 82.18 Å
Applied symmetry - Helical parameters - Δ&Phi: -0.53 °
Applied symmetry - Helical parameters - Axial symmetry: C14 (14 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 47551
Segment selectionNumber selected: 47551 / Software - Name: RELION (ver. 2.1) / Details: Number of particles seleced for reconstruction
Startup modelType of model: INSILICO MODEL
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.1)

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