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- EMDB-2534: Cryo-electron microscopy of microtubule-bound human kinesin-5 mot... -

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Basic information

Entry
Database: EMDB / ID: EMD-2534
TitleCryo-electron microscopy of microtubule-bound human kinesin-5 motor domain in the ADP.AlFx state (gold cluster in the loop5 T126C).
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 T126C
Sample
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with ADP.AlFx bound. A gold cluster is attached to the loop5 (T126C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain
Keywordscryo-electron microscopy / kinesins / microtubules / mitosis / mechanochemistry
Function / homology
Function and homology information


spindle elongation / regulation of mitotic centrosome separation / Kinesins / plus-end-directed microtubule motor activity / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / kinesin complex / spindle organization ...spindle elongation / regulation of mitotic centrosome separation / Kinesins / plus-end-directed microtubule motor activity / mitotic centrosome separation / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / microtubule motor activity / kinesin complex / spindle organization / microtubule-based movement / microtubule-based process / mitotic spindle assembly / MHC class II antigen presentation / mitotic spindle organization / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / spindle pole / spindle / microtubule cytoskeleton / mitotic cell cycle / nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / protein kinase binding / protein-containing complex / ATP binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. ...Kinesin-associated microtubule-binding domain / Kinesin-associated microtubule-binding / : / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Kinesin-like protein KIF11 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsGOULET A / MAJOR J / JUN Y / GROSS SP / ROSENFELD SR / MOORES CA
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family.
Authors: Adeline Goulet / Jennifer Major / Yonggun Jun / Steven P Gross / Steven S Rosenfeld / Carolyn A Moores /
Abstract: Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and ...Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.
History
DepositionDec 30, 2013-
Header (metadata) releaseJan 15, 2014-
Map releaseFeb 5, 2014-
UpdateFeb 12, 2014-
Current statusFeb 12, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2534.jpg

Downloads & links

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Map

FileDownload / File: emd_2534.map.gz / Format: CCP4 / Size: 245.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction 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 T126C
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 40 pix.
= 112. Å		2.8 Å/pix.
x 40 pix.
= 112. Å		2.8 Å/pix.
x 40 pix.
= 112. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.7 / Movie #1: 1.7
Minimum - Maximum-6.31151199 - 6.26682425
Average (Standard dev.)0.00195829 (±1.91305876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin7442127
Dimensions404040
Spacing404040
CellA=B=C: 112.0 Å
α=β=γ: 90.0 °

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-6.3126.2670.002

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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 attached to the loop5 (T126C).
Components
  • Sample: 13-protofilament microtubule-bound human kinesin-5 motor domain with ADP.AlFx bound. A gold cluster is attached to the loop5 (T126C).
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-5 motor domain

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Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...

SupramoleculeName: 13-protofilament microtubule-bound human kinesin-5 motor domain with ADP.AlFx bound. A gold cluster is attached to the loop5 (T126C).
type: sample / ID: 1000
Oligomeric state: 13-protofilament microtubule with one kinesin-5 motor domain bound every tubulin heterodimers
Number unique components: 3

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Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: TUBULIN ALPHA-1D CHAIN / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceUniProtKB: Tubulin alpha-1D chain / InterPro: Alpha tubulin

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: TUBULIN BETA-2B CHAIN / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: brain
SequenceUniProtKB: Tubulin beta-2B chain / InterPro: Beta tubulin, autoregulation binding site

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Macromolecule #3: Kinesin-5 motor domain

MacromoleculeName: Kinesin-5 motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: KINESIN-LIKE PROTEIN KIF11
Details: cys-lite mutant containing the substitution T126C, undecagold cluster was covalently attached to the specific cysteine residue in the loop5 (T126C)
Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Bl21 / Recombinant plasmid: pET21a
SequenceUniProtKB: Kinesin-like protein KIF11 / InterPro: Kinesin motor domain, conserved site

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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

BufferpH: 6.8 / Details: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 2 mM ADP.AlFx
GridDetails: 400 mesh holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: chamber at 24 degrees C, blot 3.5 sec

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateJul 14, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 10 / Average electron dose: 18 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected along individual microtubules.
CTF correctionDetails: FREALIGN
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: SPIDER, FREALIGN
Details: Approximately 7,300 asymmetric units were averaged in the final reconstruction.
Number images used: 561

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