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- EMDB-6188: High-resolution structures of kinesin on microtubules provide a b... -

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Basic information

Entry
Database: EMDB / ID: EMD-6188
TitleHigh-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force generation
Map dataMicrotubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
Sample
  • Sample: Microtubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
  • Protein or peptide: monomeric kinesin-1A
  • Protein or peptide: tubulin
Function / homology
Function and homology information


cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / anterograde neuronal dense core vesicle transport / anterograde dendritic transport of neurotransmitter receptor complex / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / vesicle transport along microtubule / lysosome localization ...cytoplasm organization / cytolytic granule membrane / plus-end-directed vesicle transport along microtubule / anterograde neuronal dense core vesicle transport / anterograde dendritic transport of neurotransmitter receptor complex / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / vesicle transport along microtubule / lysosome localization / positive regulation of potassium ion transport / natural killer cell mediated cytotoxicity / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Kinesins / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / plus-end-directed microtubule motor activity / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate KTN1 / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / stress granule disassembly / COPI-mediated anterograde transport / COPI-dependent Golgi-to-ER retrograde traffic / mitochondrion transport along microtubule / ciliary rootlet / centrosome localization / kinesin complex / synaptic vesicle transport / microtubule motor activity / microtubule-based movement / Insulin processing / centriolar satellite / Signaling by ALK fusions and activated point mutants / Nuclear events stimulated by ALK signaling in cancer / microtubule-based process / phagocytic vesicle / axon cytoplasm / MHC class II antigen presentation / dendrite cytoplasm / regulation of membrane potential / axon guidance / positive regulation of synaptic transmission, GABAergic / positive regulation of protein localization to plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / cellular response to type II interferon / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / vesicle / microtubule / cadherin binding / GTPase activity / protein-containing complex binding / GTP binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / membrane / identical protein binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / Kinesin motor domain signature. / 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 / Beta tubulin ...Kinesin-like protein / Kinesin motor domain signature. / 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 / 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 beta chain / Kinesin-1 heavy chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Sus scrofa (pig)
Methodhelical reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsShang ZG / Zhou KF / Xu C / Csencsits R / Cochran JC / Sindelar CV
CitationJournal: Elife / Year: 2014
Title: High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation.
Authors: Zhiguo Shang / Kaifeng Zhou / Chen Xu / Roseann Csencsits / Jared C Cochran / Charles V Sindelar /
Abstract: Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing ...Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5-6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved 'linchpin' residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's 'neck linker' element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer 'gate' each other to promote coordinated stepping along microtubules.
History
DepositionNov 18, 2014-
Header (metadata) releaseDec 3, 2014-
Map releaseDec 3, 2014-
UpdateFeb 10, 2016-
Current statusFeb 10, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j8y
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j8y
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6188.gif

Downloads & links

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Map

FileDownload / File: emd_6188.map.gz / Format: CCP4 / Size: 34.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicrotubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
Voxel sizeX=Y=Z: 2.097 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.03248612 - 0.06490891
Average (Standard dev.)-0.00070997 (±0.00664195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-159-159-54
Dimensions32032091
Spacing32032091
CellA: 671.04 Å / B: 671.04 Å / C: 190.827 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0972.0972.097
M x/y/z32032091
origin x/y/z0.0000.0000.000
length x/y/z671.040671.040190.827
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-159-159-54
NC/NR/NS32032091
D min/max/mean-0.0320.065-0.001

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Supplemental data

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Sample components

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Entire : Microtubule decorated with monomeric human kinesin (K349 construc...

EntireName: Microtubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
Components
  • Sample: Microtubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
  • Protein or peptide: monomeric kinesin-1A
  • Protein or peptide: tubulin

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Supramolecule #1000: Microtubule decorated with monomeric human kinesin (K349 construc...

SupramoleculeName: Microtubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocket.
type: sample / ID: 1000 / Details: Microtubule decorated with monomeric human kinesin
Oligomeric state: One monomer of kinesin binds to one heterodimer of tubulin
Number unique components: 2
Molecular weightTheoretical: 135 KDa

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Macromolecule #1: monomeric kinesin-1A

MacromoleculeName: monomeric kinesin-1A / type: protein_or_peptide / ID: 1 / Details: K349 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightTheoretical: 38 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pHB40p
SequenceUniProtKB: Kinesin-1 heavy chain

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Macromolecule #2: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 2 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: pig

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8 / Details: 25 mM PIPES, 25 mM NaCl, 2 mM MgCl2, 1 mM EGTA
GridDetails: 300 mesh copper grid with homemade holey carbon
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Method: No glow discharged applied; after sample application to grid, liquid was mostly 'wicked' away by edgewise application of filter paper. Subsequently, blotting and plunge freezing were ...Method: No glow discharged applied; after sample application to grid, liquid was mostly 'wicked' away by edgewise application of filter paper. Subsequently, blotting and plunge freezing were performed with a ~0.5 second delay after blotting but prior to plunging.

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 23859.4 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Details4K x 4K counting mode was used; 24 frames total were collected.
DateJun 2, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 51 / Average electron dose: 15 e/Å2
Details: Each image was collected as a stack of 24 movie frames.

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Image processing

CTF correctionDetails: done within FREALIGN
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.455 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.71 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: OTHER / Software - Name: SPIDER, FREALIGN
Details: Approximately 33,600 asymmetric units were averaged in the final reconstruction.
DetailsInitial alignment was done using customized SPIDER scripts. Reconstruction and subsequent refinement were done by FREALIGN.

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Atomic model buiding 1

Initial modelPDB ID:

4hna


Chain - #0 - Chain ID: K / Chain - #1 - Chain ID: A / Chain - #2 - Chain ID: B
SoftwareName: MDFF
DetailsMDFF was performed using explicit solvation. Side chains were removed from the MDFF target potential. Following several equilibration steps, the relative strength of the EM map potential (GSCALE term) was slowly increased from 0 to 1 over the course of 10 nanoseconds. The t = 1.2 ns time point was selected to represent the final fitted model, based on the approximate convergence of the RMSD from the starting structure.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Target criteria: RMSD from the starting structure was monitored for convergence
Output model

PDB-3j8y:
High-resolution structure of ATP analog-bound kinesin on microtubules

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