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- PDB-4etp: C-terminal motor and motor homology domain of Kar3Vik1 fused to a... -

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Basic information

Entry
Database: PDB / ID: 4etp
TitleC-terminal motor and motor homology domain of Kar3Vik1 fused to a synthetic heterodimeric coiled coil
Components
  • Kinesin-like protein KAR3
  • Spindle pole body-associated protein VIK1
KeywordsMOTOR PROTEIN / Kinesin Motor Protein / Kinesin Motor Homology Domain / karyogamy / mitosis / microtubules / internal Vik1 crosslink with N / N-Ethylenebis(iodoacetamide) / Nucleus
Function / homology
Function and homology information


minus-end-directed kinesin ATPase / microtubule bundle formation involved in mitotic spindle midzone assembly / nuclear migration involved in conjugation with cellular fusion / protein transport along microtubule to mitotic spindle pole body / karyogamy involved in conjugation with cellular fusion / cytoskeletal anchor activity / minus-end-directed microtubule motor activity / spindle pole body / mitotic sister chromatid cohesion / kinesin complex ...minus-end-directed kinesin ATPase / microtubule bundle formation involved in mitotic spindle midzone assembly / nuclear migration involved in conjugation with cellular fusion / protein transport along microtubule to mitotic spindle pole body / karyogamy involved in conjugation with cellular fusion / cytoskeletal anchor activity / minus-end-directed microtubule motor activity / spindle pole body / mitotic sister chromatid cohesion / kinesin complex / cytoplasmic microtubule / microtubule-based process / regulation of mitotic spindle organization / isomerase activity / meiotic cell cycle / spindle pole / mitotic cell cycle / chromosome / microtubule binding / microtubule / cell division / ATP hydrolysis activity / ATP binding / nucleus / cytoplasm
Similarity search - Function
Kinesin - #20 / Spindle pole body-associated protein Vik1/Cik1, microtubule binding domain / Microtubule binding / Kinesin motor domain / Kinesin / Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. ...Kinesin - #20 / Spindle pole body-associated protein Vik1/Cik1, microtubule binding domain / Microtubule binding / Kinesin motor domain / Kinesin / 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 / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / N,N'-ethane-1,2-diylbis(2-iodoacetamide) / Kinesin-like protein KAR3 / Spindle pole body-associated protein VIK1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsRank, K.C. / Chen, C.J. / Cope, J. / Porche, K. / Hoenger, A. / Gilbert, S.P. / Rayment, I.
CitationJournal: J Cell Biol / Year: 2012
Title: Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.
Authors: Katherine C Rank / Chun Ju Chen / Julia Cope / Ken Porche / Andreas Hoenger / Susan P Gilbert / Ivan Rayment /
Abstract: Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the ...Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.
History
DepositionApr 24, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 27, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2013Group: Database references
Revision 1.2Sep 13, 2023Group: 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_conn_type / 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 / _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.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _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_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_conn_type.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

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Kinesin-like protein KAR3
B: Spindle pole body-associated protein VIK1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,05811
Polymers83,6582
Non-polymers1,4009
Water4,432246
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5340 Å2
ΔGint-47 kcal/mol
Surface area34440 Å2
MethodPISA
Unit cell
Length a, b, c (Å)88.060, 94.939, 114.590
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Kinesin-like protein KAR3 / Nuclear fusion protein


Mass: 45178.941 Da / Num. of mol.: 1 / Fragment: Kar3 (unp residues 352-729) / Mutation: C391L, C469A, C517A, C566V, C655V)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: KAR3, kar3p, P9659.16, YPR141C / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P17119
#2: Protein Spindle pole body-associated protein VIK1 / Vegetative interaction with KAR3 protein 1


Mass: 38478.742 Da / Num. of mol.: 1 / Fragment: Vik1 (unp residues 341-647) / Mutation: C377V, C436A, C536A, C596A, C640A, E355C, K423C)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: VIK1, Vik1p, YPL253C / Plasmid: pKLD37 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q12045

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Non-polymers , 5 types, 255 molecules

#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-EBC / N,N'-ethane-1,2-diylbis(2-iodoacetamide)


Mass: 395.965 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H10I2N2O2
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 246 / Source method: isolated from a natural source / Formula: H2O

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Details

Nonpolymer detailsTHE STARTING MATERIAL USED FOR CROSS LINKING CONTAINED A IODINE ATOM THAT HAS LEAVED UPON REACTION ...THE STARTING MATERIAL USED FOR CROSS LINKING CONTAINED A IODINE ATOM THAT HAS LEAVED UPON REACTION WITH CYS. THE FINAL PRODUCT CORRESPONDS TO THE LIGAND EBC LINKED TO THE SIDE CHAINS OF CYS
Sequence detailsNATIVE KAR3VIK1 CONTAINS EXTENSIVE BUT WEAK COILED-COIL. FOR CRYSTALLIZATION, ALL BUT THE LAST C- ...NATIVE KAR3VIK1 CONTAINS EXTENSIVE BUT WEAK COILED-COIL. FOR CRYSTALLIZATION, ALL BUT THE LAST C-TERMINAL 2.5 NATIVE HEPTADS WERE REPLACED WITH STABILIZING HETERODIMERIC SYNTHETIC COILED-COIL. THIS FUSION DOES NOT AFFECT PROTEIN ACTIVITY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.86 Å3/Da / Density % sol: 57.04 %
Crystal growTemperature: 277 K / Method: evaporation / pH: 7.5
Details: 16% monomethyl polyethylene glycol 2000, 100 mM Hepes, 50 mM sodium citrate, Batch, pH 7.5, EVAPORATION, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.979206 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Dec 2, 2010
RadiationMonochromator: Rosenbaum-Rock high-resolution double-crystal monochromator. LN2 cooled first crystal, sagittal focusing 2nd crystal
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979206 Å / Relative weight: 1
ReflectionResolution: 2.3→25 Å / Num. all: 43354 / Num. obs: 43179 / % possible obs: 99.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 8.6 % / Rmerge(I) obs: 0.051 / Rsym value: 0.069 / Net I/σ(I): 19.7
Reflection shellResolution: 2.3→2.34 Å / Redundancy: 6.2 % / Rmerge(I) obs: 0.601 / Mean I/σ(I) obs: 3.2 / Num. unique all: 2701 / Rsym value: 0.593 / % possible all: 99.8

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Processing

Software
NameVersionClassification
HKL-2000data collection
PHASERphasing
REFMAC5.6.0117refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 3KAR and 2O0A

3kar

2o0a


Resolution: 2.3→24.97 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.936 / SU B: 10.825 / SU ML: 0.142 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.274 / ESU R Free: 0.215 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.24472 2156 5 %RANDOM
Rwork0.20731 ---
all0.20923 42866 --
obs0.20923 42866 98.96 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1 Å / Solvent model: MASK
Displacement parametersBiso mean: 69.055 Å2
Baniso -1Baniso -2Baniso -3
1-1.16 Å2-0 Å20 Å2
2--0.4 Å20 Å2
3----1.56 Å2
Refinement stepCycle: LAST / Resolution: 2.3→24.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5344 0 74 246 5664
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.025510
X-RAY DIFFRACTIONr_angle_refined_deg0.8911.9737421
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.5115662
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.49825.573262
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.006151043
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.4281523
X-RAY DIFFRACTIONr_chiral_restr0.0560.2865
X-RAY DIFFRACTIONr_gen_planes_refined0.0020.024000
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.301→2.361 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.33 135 -
Rwork0.269 2701 -
obs--96.14 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7456-0.61740.02270.92640.25670.3908-0.0576-0.0486-0.00050.02210.1274-0.01010.0213-0.0222-0.06970.0949-0.0227-0.01480.14160.00660.13993.044-18.868-6.973
21.36961.06120.16052.8576-0.29231.8143-0.1920.3489-0.0442-0.91760.3067-0.2479-0.33470.4166-0.11460.5268-0.1720.15810.2445-0.07940.059923.463-26.923-42.563
39.6682-2.1469-2.285.1871-2.66385.075-0.6874-0.5001-1.1764-0.89210.0893-0.03781.51920.0140.59820.73280.05670.15980.04040.05190.181423.616-75.444-31.233
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A353 - 723
2X-RAY DIFFRACTION2B341 - 638
3X-RAY DIFFRACTION2B701
4X-RAY DIFFRACTION3A331 - 352
5X-RAY DIFFRACTION3B320 - 340

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