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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 chainKinesin
  • 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 / 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 / regulation of membrane potential / dendrite cytoplasm / 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
ADENOSINE-5'-TRIPHOSPHATE / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Kinesin-1 heavy chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5 Å
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 /
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 20, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 10, 2014Provider: repository / Type: Initial release
Revision 1.1Jun 3, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-6188
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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
Polymers139,4263
Non-polymers1,4984
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 8.5215 Å / Rotation per n subunits: -25.77 °)
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 , 3 types, 3 molecules KAB

#1: Protein Kinesin-1 heavy chain / Kinesin / 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 method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF5B, KNS, KNS1 / Production host: Escherichia coli (E. coli) / References: UniProt: P33176
#2: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


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


Mass: 49983.824 Da / Num. of mol.: 1 / Source method: isolated from a natural source / 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 / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#7: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D 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-tubulinTubulin1
Molecular weightValue: 0.135 MDa / Experimental value: NO
Buffer solutionName: 25 mM PIPES, 25 mM NaCl, 2 mM MgCl2, 1 mM EGTA / pH: 6.8 / Details: 25 mM PIPES, 25 mM NaCl, 2 mM MgCl2, 1 mM EGTA
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 ...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 ...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

MicroscopyModel: 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 X / 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 scansNum. digital images: 51
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (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 ° / 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 / Num. of particles: 49961 / Nominal pixel size: 2.097 Å / Actual pixel size: 2.097 Å
Details: Initial alignment was done using customized SPIDER scripts. Reconstruction and subsequent refinement were done by FREALIGN.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Target criteria: RMSD from the starting structure was monitored for convergence.
Details: REFINEMENT PROTOCOL--flexible DETAILS--MDFF was performed using explicit solvation, after placing active-site water coordinates identified in high-resolution crystal structures of kinesins ...Details: 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.
Atomic model building

3D fitting-ID: 1 / Accession code: 4HNA / Initial refinement model-ID: 1 / PDB-ID: 4HNA

4hna

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1K
2A
3B
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms9343 0 92 0 9435

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