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- EMDB-2077: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -

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Entry
Database: EMDB / ID: 2077
TitleElectron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in AMPPNP state.
Keywordscancer / electron cryo-microscopy / kinesin / microtubule / mitosis
Sample13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP
SourceBos taurus / mammal / Cattle / ウシ /
Homo sapiens / human
Map data3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site
Methodsingle particle reconstruction, at 9.7 Å resolution
AuthorsGoulet A / Behnke-Parks WM / Sindelar C / Rosenfeld S / Moores C
CitationJ. Biol. Chem., 2012, 287, 44654-44666

J. Biol. Chem., 2012, 287, 44654-44666 Yorodumi Papers
The structural basis of force generation by the mitotic motor kinesin-5.
Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores

Validation ReportPDB-ID: 4aqv

SummaryFull reportAbout validation report
DateDeposition: Apr 19, 2012 / Header (metadata) release: May 17, 2012 / Map release: Nov 21, 2012 / Last update: Jan 9, 2013

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Structure visualization

Movie
  • Colored surface
  • Surface level: 1.1
  • Imaged by UCSF CHIMERA
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  • Surface view colored by cylindrical radius
  • Surface level: 1.1
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-4aqv
  • Surface level: 1.1
  • Imaged by UCSF CHIMERA
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4aqv
  • Imaged by Jmol
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Supplemental images

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Map

Fileemd_2077.map.gz (map file in CCP4 format, 357 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
45 pix
2.8 Å/pix.
= 126. Å
45 pix
2.8 Å/pix.
= 126. Å
45 pix
2.8 Å/pix.
= 126. 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:1.1 (by author), 1.1 (movie #1):
Minimum - Maximum-6.57703495 - 8.06319046
Average (Standard dev.)0.08032296 (1.81039548)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions454545
Origin000
Limit444444
Spacing454545
CellA=B=C: 126 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z454545
origin x/y/z0.0000.0000.000
length x/y/z126.000126.000126.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS454545
D min/max/mean-6.5778.0630.080

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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 AMPPNP
Number of components: 2
Oligomeric State: 13-protofilament microtubule with one kineisn-5 motor domain bound every tubulin heterodimers

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Component #1: protein, alpha-beta tubulin dimer

ProteinName: alpha-beta tubulin dimer / Oligomeric Details: heterodimer / Recombinant expression: No
SourceSpecies: Bos taurus / mammal / Cattle / ウシ /
Source (natural)Organ or tissue: brain

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Component #2: protein, Kinesin-5 motor domain

ProteinName: Kinesin-5 motor domain / Oligomeric Details: monomer / 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

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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, 5mM AMPPNP
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 2.5 sec

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Jan 10, 2011
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: 700 - 2200 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: 46 / Scanner: ZEISS SCAI / Sampling size: 7 microns / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 3587
Details: The particles were selected along individual microtubules.
3D reconstructionSoftware: SPIDER, FREALIGN / CTF correction: FREALIGN
Details: Approximately 50,000 asymmetric units were averaged in the final reconstruction.
Resolution: 9.7 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera, FlexEM / Refinement protocol: flexible / Target criteria: cross-correlation / Refinement space: REAL
Details: Protocol: rigid body then flexible fitting. The domain was fitted as a rigid body. The N-terminal residues 6 to 16 were built in the EM map and the final model was refined by flexible fitting.
Input PDB model: 3HQD
Chain ID: A
Modeling #2Software: Chimera / Refinement protocol: rigid body / Target criteria: cross-correlation / Refinement space: REAL
Details: Protocol: rigid body. alpha- and b-tubulin were separately fitted.
Input PDB model: 1JFF
Chain ID: A, B
Output model

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