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- EMDB-6187: High-resolution structures of kinesin on microtubules provide a b... -

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Basic information

Entry
Database: EMDB / ID: EMD-6187
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 an empty nucleotide pocket.
Sample
  • Sample: Microtubule decorated with monomeric human kinesin (K349 construct) having an empty nucleotide pocket.
  • Protein or peptide: monomeric kinesin-1A
  • Protein or peptide: tubulin
Keywordsmolecular motors / kinesin / myosin / microtubules / cytoskeletal motors
Function / homology
Function and homology information


regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet ...regulation of modification of synapse structure, modulating synaptic transmission / plus-end-directed vesicle transport along microtubule / cytoplasm organization / cytolytic granule membrane / anterograde dendritic transport of neurotransmitter receptor complex / anterograde neuronal dense core vesicle transport / mitocytosis / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / ciliary rootlet / lysosome localization / positive regulation of potassium ion transport / plus-end-directed microtubule motor activity / vesicle transport along microtubule / RHO GTPases activate KTN1 / Kinesins / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / kinesin complex / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / microtubule motor activity / Recruitment of NuMA to mitotic centrosomes / centrosome localization / COPI-mediated anterograde transport / mitochondrion transport along microtubule / COPI-dependent Golgi-to-ER retrograde traffic / microtubule-based movement / stress granule disassembly / natural killer cell mediated cytotoxicity / Insulin processing / synaptic vesicle transport / postsynaptic cytosol / microtubule-based process / phagocytic vesicle / axon cytoplasm / MHC class II antigen presentation / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / regulation of membrane potential / positive regulation of protein localization to plasma membrane / cellular response to type II interferon / structural constituent of cytoskeleton / centriolar satellite / microtubule cytoskeleton organization / Signaling by ALK fusions and activated point mutants / mitotic cell cycle / microtubule cytoskeleton / nuclear membrane / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / vesicle / microtubule / cadherin binding / GTPase activity / GTP binding / protein-containing complex binding / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / membrane / cytoplasm / cytosol
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) releaseNov 26, 2014-
Map releaseNov 26, 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: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-3j8x
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j8x
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6187.gif

Downloads & links

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Map

FileDownload / File: emd_6187.map.gz / Format: CCP4 / Size: 23.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicrotubule decorated with monomeric human kinesin (K349 construct) having an empty nucleotide pocket.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.99 Å/pix.
x 96 pix.
= 191.04 Å		1.99 Å/pix.
x 256 pix.
= 509.44 Å		1.99 Å/pix.
x 256 pix.
= 509.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.99 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02259131 - 0.05996572
Average (Standard dev.)0.00012049 (±0.00745614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-58
Dimensions25625696
Spacing25625696
CellA: 509.44 Å / B: 509.44 Å / C: 191.04001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.991.991.99
M x/y/z25625696
origin x/y/z0.0000.0000.000
length x/y/z509.440509.440191.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-58
NC/NR/NS25625696
D min/max/mean-0.0230.0600.000

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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 an empty nucleotide pocket.
Components
  • Sample: Microtubule decorated with monomeric human kinesin (K349 construct) having an empty 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 an empty nucleotide pocket.
type: sample / ID: 1000
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

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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

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Electron microscopy

MicroscopeFEI TECNAI F30
Details8K x 8K Super-resolution mode was used; 10 frames total were collected.
DateApr 15, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsInitial alignment was done using customized SPIDER scripts. Reconstruction and subsequent refinement was done by FREALIGN.
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.455 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.77 °
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 140,000 asymmetric units were averaged in the final reconstruction.
CTF correctionDetails: done within 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, after placing active-site water coordinates identified in high-resolution crystal structures of kinesin's ATP-like state. 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 = 1.4 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-3j8x:
High-resolution structure of no-nucleotide kinesin on microtubules

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