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- EMDB-2535: cryo-electron microscopy of microtubule-bound human kinesin-5 mot... -

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Entry
Database: EMDB / ID: 2535
Titlecryo-electron microscopy of microtubule-bound human kinesin-5 motor domain in the ADP.AlFx state (gold cluster in the neck linker V365C).
Keywordscryo-electron microscopy / kinesins / microtubules / mitosis / mechanochemistry
Sample13-protofilament microtubule-bound human kinesin-5 motor domain with ADP.AlFx bound. A gold cluster is covalently attached to the neck linker (V365C).
SourceBos taurus / mammal / Cattle / ウシ /
Homo sapiens / human
Map dataReconstruction of microtubule-bound human kinesin-5 motor domain in presence of the ATP analog ADP.AlFx and with a glod cluster covalently attached to the residue V365C
Methodsingle particle reconstruction, at 16 Å resolution
AuthorsGOULET A MAJOR J JUN Y / GROSS SP / ROSENFELD SR / MOORES CA
CitationProc. Natl. Acad. Sci. U.S.A., 2014, 111, 1837-1842

Proc. Natl. Acad. Sci. U.S.A., 2014, 111, 1837-1842 Yorodumi Papers
Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family.
Adeline Goulet / Jennifer Major / Yonggun Jun / Steven P Gross / Steven S Rosenfeld / Carolyn A Moores

DateDeposition: Dec 30, 2013 / Header (metadata) release: Jan 15, 2014 / Map release: Feb 5, 2014 / Last update: Feb 19, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.05
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by height
  • Surface level: 1.05
  • Imaged by UCSF CHIMERA
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Supplemental images

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Map

Fileemd_2535.map.gz (map file in CCP4 format, 251 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
40 pix
2.8 Å/pix.
= 112. Å
40 pix
2.8 Å/pix.
= 112. Å
40 pix
2.8 Å/pix.
= 112. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 2.8 Å
Density
Contour Level:1.05 (by author), 1.05 (movie #1):
Minimum - Maximum-6.29903316 - 7.50455666
Average (Standard dev.)-0.00828098 (1.95693755)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions404040
Origin7442127
Limit11381166
Spacing404040
CellA=B=C: 112 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z404040
origin x/y/z0.0000.0000.000
length x/y/z112.000112.000112.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS4274127
NC/NR/NS404040
D min/max/mean-6.2997.505-0.008

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Supplemental data

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Sample components

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Entire 13-protofilament microtubule-bound human kinesin-5 motor domain w...

EntireName: 13-protofilament microtubule-bound human kinesin-5 motor domain with ADP.AlFx bound. A gold cluster is covalently attached to the neck linker (V365C).
Number of components: 3
Oligomeric State: 13-protofilament microtubule with one kinesin-5 motor domain bound every tubulin heterodimers

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Component #1: protein, alpha tubulin

ProteinName: alpha tubulin / a.k.a: TUBULIN ALPHA-1D CHAIN / Recombinant expression: No
SourceSpecies: Bos taurus / mammal / Cattle / ウシ /
Source (natural)Organ or tissue: brain
External referencesUniProt: UniProt: Q2HJ86 / InterPro: InterPro: 002452

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Component #2: protein, beta tubulin

ProteinName: beta tubulin / a.k.a: TUBULIN BETA-2B CHAIN / Recombinant expression: No
SourceSpecies: Bos taurus / mammal / Cattle / ウシ /
Source (natural)Organ or tissue: brain
External referencesInterPro: InterPro: 013838 / UniProt: UniProt: Q6B856

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Component #3: protein, Kinesin-5 motor domain

ProteinName: Kinesin-5 motor domain / a.k.a: KINESIN-LIKE PROTEIN KIF11
Details: cys-lite mutant containing the substitution V365C, undecagold cluster was attached to the specific cysteine residue in the neck linker (V365C)
Recombinant expression: Yes
SourceSpecies: Homo sapiens / human
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Vector: pET21a / Strain: Bl21
External referencesUniProt: UniProt: P52732 / InterPro: InterPro: 019821

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionBuffer solution: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5 mM ADP.AlFx
pH: 6.8
Support film400 mesh holey carbon grids
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Method: chamber at 24 degrees C, blot 3.5 sec

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Oct 13, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 18 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1100 - 2600 nm
Specimen HolderModel: GATAN LIQUID NITROGEN / Temperature: 90 K
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 10 / Scanner: ZEISS SCAI / Sampling size: 7 microns / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 10000
Details: The particles were selected along individual microtubules.
3D reconstructionSoftware: SPIDER, FREALIGN / CTF correction: FREALIGN
Details: Approximately 13,000 asymmetric units were averaged in the final reconstruction.
Resolution: 16 Å / Resolution method: FSC 0.5, semi-independent

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