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- PDB-3j6h: Nucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule -

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Basic information

Entry
Database: PDB / ID: 3j6h
TitleNucleotide-free Kinesin motor domain complexed with GMPCPP-microtubule
Components
  • Kinesin heavy chain isoform 5C
  • Tubulin alpha-1A chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN/MOTOR PROTEIN / Kinesin / Motor domain / Rigor-conformation / Nucleotide-free kinesin / Microtubule / GMPCPP-microtubule / tubulin / Axonal transport / STRUCTURAL PROTEIN-MOTOR PROTEIN complex
Function / homology
Function and homology information


distal axon / anterograde dendritic transport of messenger ribonucleoprotein complex / anterograde dendritic transport of neurotransmitter receptor complex / anterograde axonal protein transport / intracellular mRNA localization / apolipoprotein receptor binding / plus-end-directed microtubule motor activity / motor neuron axon guidance / ciliary rootlet / postsynaptic cytosol ...distal axon / anterograde dendritic transport of messenger ribonucleoprotein complex / anterograde dendritic transport of neurotransmitter receptor complex / anterograde axonal protein transport / intracellular mRNA localization / apolipoprotein receptor binding / plus-end-directed microtubule motor activity / motor neuron axon guidance / ciliary rootlet / postsynaptic cytosol / kinesin complex / synaptic vesicle transport / microtubule motor activity / mRNA transport / axonal growth cone / axon cytoplasm / dendrite cytoplasm / axon guidance / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule binding / microtubule / hydrolase activity / neuron projection / GTPase activity / dendrite / neuronal cell body / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1A chain / Tubulin beta chain / Kinesin heavy chain isoform 5C
Similarity search - Component
Biological speciesMus musculus (house mouse)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsMorikawa, M. / Yajima, H. / Nitta, R. / Inoue, S. / Ogura, T. / Sato, C. / Hirokawa, N.
CitationJournal: EMBO J / Year: 2015
Title: X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding.
Authors: Manatsu Morikawa / Hiroaki Yajima / Ryo Nitta / Shigeyuki Inoue / Toshihiko Ogura / Chikara Sato / Nobutaka Hirokawa /
Abstract: The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state ...The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.
History
DepositionFeb 21, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Apr 1, 2015Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Data collection / Database references
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_software / struct_ref_seq_dif
Item: _em_software.image_processing_id / _em_software.name / _struct_ref_seq_dif.details
Revision 1.2Mar 20, 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_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_asym_id / _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.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_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.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • EMDB-5916
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1A chain
B: Tubulin beta chain
K: Kinesin heavy chain isoform 5C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,0518
Polymers135,8623
Non-polymers1,1895
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules ABK

#1: Protein Tubulin alpha-1A chain / / Alpha-tubulin 1 / Tubulin alpha-1 chain


Mass: 48436.625 Da / Num. of mol.: 1 / Fragment: UNP residues 2-437 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: brain / References: UniProt: P02550
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 47809.746 Da / Num. of mol.: 1 / Fragment: UNP residues 2-427 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: brain / References: UniProt: P02554
#3: Protein Kinesin heavy chain isoform 5C / KIF5C / Kinesin heavy chain neuron-specific 2


Mass: 39615.137 Da / Num. of mol.: 1 / Fragment: UNP residues 1-345
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kif5c / Plasmid: pET21b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P28738

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: 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
#6: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#7: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4

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Details

Sequence detailsTHIS SEQUENCE IS NATURAL VARIANT.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Kinesin motor domain complexed with GMPCPP-microtubule
Type: COMPLEX
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA KF80 / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL 2010F / Date: Jan 1, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 40000 X / Calibrated magnification: 40000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1200 nm / Cs: 3.3 mm
Specimen holderTemperature: 100 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 10 e/Å2 / Film or detector model: KODAK SO-163 FILM
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1MODELLERmodel fitting
2UCSF Chimeramodel fitting
3IMAGIC53D reconstruction
4MATLAB3D reconstruction
CTF correctionDetails: Each filament
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Single ParticleSingle particle analysis / Resolution: 8.1 Å / Num. of particles: 302000 / Nominal pixel size: 2.5 Å / Actual pixel size: 2.5 Å / Num. of class averages: 18 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Target criteria: Cross-correlation, Average map value, Atoms inside the contour
Details: METHOD--Local refinement, Domain fitting REFINEMENT PROTOCOL--Rigid body refinement DETAILS--Initial local fitting was done using Chimera and for some loops Modeller was used.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11JFF

1jff

11JFF1PDBexperimental model
21JFF

1jff

11JFF1PDBexperimental model
33WRD

3wrd

13WRD2PDBexperimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms8739 0 71 0 8810

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