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- EMDB-2771: Conserved mechanisms of microtubule-stimulated ADP release, ATP b... -

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Basic information

Entry
Database: EMDB / ID: EMD-2771
TitleConserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins
Map data13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
Sample
  • Sample: 13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-1 motor domain
Keywordskinesin / microtubule / cryo-EM / cryo-electron microscopy
Function / homology
Function and homology information


plus-end-directed kinesin ATPase / anterograde dendritic transport of neurotransmitter receptor complex / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet / plus-end-directed microtubule motor activity / RHO GTPases activate KTN1 / Kinesins / positive regulation of axon guidance / kinesin complex ...plus-end-directed kinesin ATPase / anterograde dendritic transport of neurotransmitter receptor complex / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet / plus-end-directed microtubule motor activity / RHO GTPases activate KTN1 / Kinesins / positive regulation of axon guidance / kinesin complex / microtubule motor activity / COPI-dependent Golgi-to-ER retrograde traffic / microtubule-based movement / Insulin processing / synaptic vesicle transport / cytoskeletal motor activity / kinesin binding / postsynaptic cytosol / microtubule-based process / cytoplasmic microtubule / vesicle-mediated transport / axon cytoplasm / MHC class II antigen presentation / cellular response to interleukin-4 / dendrite cytoplasm / axon guidance / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / double-stranded RNA binding / microtubule cytoskeleton / perikaryon / microtubule binding / chemical synaptic transmission / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cilium / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / GTP binding / perinuclear region of cytoplasm / ATP hydrolysis activity / ATP binding / metal ion binding / membrane / cytoplasm / cytosol
Similarity search - Function
Kinesin-like protein / 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 ...Kinesin-like protein / 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
Tubulin alpha-1B chain / Kinesin heavy chain isoform 5A / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.7 Å
AuthorsAtherton J / Farabella I / Yu IM / Rosenfeld SS / Houdusse A / Topf M / Moores C
CitationJournal: Elife / Year: 2014
Title: Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.
Authors: Joseph Atherton / Irene Farabella / I-Mei Yu / Steven S Rosenfeld / Anne Houdusse / Maya Topf / Carolyn A Moores /
Abstract: Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation ...Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.
History
DepositionAug 27, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseSep 24, 2014-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uy0
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4uy0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2771_1.jpg

Downloads & links

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Map

FileDownload / File: emd_2771.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 400 pix.
= 600. Å		1.5 Å/pix.
x 400 pix.
= 600. Å		1.5 Å/pix.
x 400 pix.
= 600. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.3 / Movie #1: 2.3
Minimum - Maximum-6.65302849 - 10.887493129999999
Average (Standard dev.)0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 600.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z600.000600.000600.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-6.65310.8870.000

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Supplemental data

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Sample components

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Entire : 13-protofilament microtubule-bound human kinesin-1 motor domain w...

EntireName: 13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
Components
  • Sample: 13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
  • Protein or peptide: alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Kinesin-1 motor domain

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Supramolecule #1000: 13-protofilament microtubule-bound human kinesin-1 motor domain w...

SupramoleculeName: 13-protofilament microtubule-bound human kinesin-1 motor domain with ADPAlFx
type: sample / ID: 1000
Oligomeric state: A kinesin motor domain binds to each alpha-beta tubulin heterodimer
Number unique components: 3

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Macromolecule #1: alpha tubulin

MacromoleculeName: alpha tubulin / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: Bovine
SequenceInterPro: Alpha tubulin

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / Oligomeric state: heterodimer / Recombinant expression: No
Source (natural)Organism: Bos taurus (domestic cattle) / synonym: Bovine
SequenceInterPro: Beta tubulin, autoregulation binding site

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Macromolecule #3: Kinesin-1 motor domain

MacromoleculeName: Kinesin-1 motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: Kif5A, Kin1 / Details: with bound ADPAlFx / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET151-D-TOPO
SequenceInterPro: 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: 20mM PIPES, 2mM MgCl2, 1mM EGTA, 2mM DTT, 2mM ADPAlFx
GridDetails: 400 mesh holey carbon grids, air glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I
Method: Chamber at 24 degrees C, add microtubule droplet, blot 0.5 sec, add kinesin motor domain droplet, blot 3.5s.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150000 times magnification
DateDec 10, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 149 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.4 µm
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected interactively at the computer terminal
CTF correctionDetails: Frealign
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.7 Å / Resolution method: OTHER / Software - Name: Spider, Frealign
Details: Pseudo-symmetry was utilised to generate the asymmetric unit at improved resolution
Number images used: 65572

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Atomic model buiding 1

Initial modelPDB ID:

4hna


Chain - Chain ID: K
SoftwareName: Chimera, Flex-EM
DetailsInitial model based on 4HNA rigid fitted in Chimera, then flexible fitting performed in Flex-EM (Topf et al., 2008)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross correlation
Output model

PDB-4uy0:
Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

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