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

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Basic information

Entry
Database: PDB / ID: 3j8y
TitleHigh-resolution structure of ATP analog-bound kinesin on microtubules
Components
  • Kinesin-1 heavy chain
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN/STRUCTURAL PROTEIN / molecular motors / kinesin / myosin / microtubules / cytoskeletal motors / MOTOR PROTEIN-STRUCTURAL PROTEIN complex
Function / homologyKinesin motor domain / Kinesin-like protein / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Tubulin / Regulation of PLK1 Activity at G2/M Transition / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal ...Kinesin motor domain / Kinesin-like protein / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Tubulin / Regulation of PLK1 Activity at G2/M Transition / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Kinesin motor domain, conserved site / Tubulin, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Tubulin/FtsZ, GTPase domain superfamily / Recruitment of mitotic centrosome proteins and complexes / Kinesin motor domain profile. / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / RHO GTPases activate KTN1 / Insulin processing / MHC class II antigen presentation / Kinesin motor domain signature. / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin C-terminal domain / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Loss of Nlp from mitotic centrosomes / Separation of Sister Chromatids / Loss of proteins required for interphase microtubule organization from the centrosome / Recycling pathway of L1 / Kinesins / Carboxyterminal post-translational modifications of tubulin / AURKA Activation by TPX2 / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases Activate Formins / RHO GTPases activate IQGAPs / Intraflagellar transport / Anchoring of the basal body to the plasma membrane / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / positive regulation of voltage-gated sodium channel activity / cytoplasm organization / plus-end-directed vesicle transport along microtubule / anterograde neuronal dense core vesicle transport / retrograde neuronal dense core vesicle transport / anterograde axonal protein transport / vesicle transport along microtubule / positive regulation of potassium ion transport / positive regulation of vesicle fusion / microtubule lateral binding / positive regulation of intracellular protein transport / stress granule disassembly / JUN kinase binding / regulation of synapse organization / ciliary rootlet / centrosome localization / spindle assembly / kinesin complex / MHC class I protein binding / microtubule motor activity / nuclear envelope lumen / positive regulation of insulin secretion involved in cellular response to glucose stimulus / axon cytoplasm / microtubule-based movement / microtubule-based process / phagocytic vesicle / regulation of membrane potential / axonal growth cone / cytoplasmic ribonucleoprotein granule / cellular response to interferon-gamma / structural constituent of cytoskeleton / microtubule cytoskeleton organization / hippocampus development / neuron migration / positive regulation of synaptic transmission, GABAergic / microtubule organizing center / positive regulation of protein localization to plasma membrane / cell body / mitotic cell cycle / microtubule / microtubule binding / vesicle / ATPase activity / cadherin binding / GTPase activity / GTP binding / ubiquitin protein ligase binding / perinuclear region of cytoplasm
Function and homology information
Specimen sourceHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 5 Å resolution
AuthorsShang, Z. / Zhou, K. / Xu, C. / Csencsits, R. / Cochran, J.C. / Sindelar, C.V.
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 20, 2014 / Release: Dec 10, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 10, 2014Structure modelrepositoryInitial release
1.1Jun 3, 2015Structure modelDatabase references
1.2Jul 18, 2018Structure modelAuthor supporting evidence / Data collectionem_single_particle_entity / em_software_em_software.image_processing_id / _em_software.name

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Assembly

Deposited unit
K: Kinesin-1 heavy chain
A: Tubulin alpha-1B chain
B: Tubulin beta-2B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,9247
Polyers139,4263
Non-polymers1,4984
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Helical symmetryCircular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Number of operations: 1 / Rise per n subunits: 8.5215 Å / Rotation per n subunits: -25.77 deg.
DetailsThe reconstructed 14-protofilament microtubule is pseudo-symmetric, containing a seam with 3 starts per tubulin monomer, or 1.5 starts per tubulin dimer.

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Components

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Protein/peptide , 3 types, 3 molecules KAB

#1: Protein/peptide Kinesin-1 heavy chain / / Conventional kinesin heavy chain / Ubiquitous kinesin heavy chain / UKHC


Mass: 39238.145 Da / Num. of mol.: 1
Fragment: Truncated catalytic head domain (monomeric, UNP residues 1-349)
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF5B, KNS, KNS1 / Production host: Escherichia coli (E. coli) / References: UniProt: P33176
#2: Protein/peptide Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 1 / Source: (natural) Sus scrofa (pig) / References: UniProt: Q2XVP4
#3: Protein/peptide Tubulin beta-2B chain


Mass: 49983.824 Da / Num. of mol.: 1 / Source: (natural) Sus scrofa (pig) / References: UniProt: F2Z5B2

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#7: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 1 / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent ID
1Microtubule decorated with monomeric human kinesin (K349 construct) having ADP aluminum fluoride complex bound in the nucleotide pocketCOMPLEXOne monomer of kinesin binds to one heterodimer of tubulin.0
2Human monomeric kinesin-1A construct1
3Alpha-tubulin1
4Beta-tubulin1
Molecular weightValue: 0.135 MDa / Experimental value: NO
Buffer solutionName: 25 mM PIPES, 25 mM NaCl, 2 mM MgCl2, 1 mM EGTA / Details: 25 mM PIPES, 25 mM NaCl, 2 mM MgCl2, 1 mM EGTA / pH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 300 mesh copper grid with homemade holey carbon
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: No glow discharge was 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 ~0.5 second delay after blotting but prior to plunging into liquid ethane.
Method: No glow discharge was 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 ~0.5 second delay after blotting but prior to plunging.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TITAN / Date: Jun 2, 2013
Details: 4K x 4K counting mode was used. 24 frames total were collected.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 23859 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNumber digital images: 51
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2FREALIGN3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: done within FREALIGN
Helical symmertyAngular rotation/subunit: 25.77 deg. / Axial rise/subunit: 8.5215 Å / Axial symmetry: C1
Details: The reconstructed 14-protofilament microtubule is pseudo-symmetric, containing a seam with 3 starts per tubulin monomer, or 1.5 starts per tubulin dimer.
3D reconstructionMethod: Single particleSingle particle analysis / Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 49961 / Nominal pixel size: 2.097 / Actual pixel size: 2.097
Details: Initial alignment was done using customized SPIDER scripts. Reconstruction and subsequent refinement were done by FREALIGN.
Symmetry type: HELICAL
Atomic model buildingDetails: REFINEMENT PROTOCOL--flexible DETAILS--MDFF was performed using explicit solvation, after placing active-site water coordinates identified in high-resolution crystal structures of kinesins ATP-like state. Side chains were excluded 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.
Ref protocol: FLEXIBLE FIT / Ref space: REAL
Target criteria: RMSD from the starting structure was monitored for convergence.
Atomic model building
IDPDB-IDPdb chain ID 3D fitting ID
14HNAK1
24HNAA1
34HNAB1
Number of atoms included #LASTProtein: 9343 / Nucleic acid: 0 / Ligand: 92 / Solvent: 0 / Total: 9435

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