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- PDB-7lff: Crystal structure of the Candida albicans kinesin-8 motor domain -

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Basic information

Entry
Database: PDB / ID: 7lff
TitleCrystal structure of the Candida albicans kinesin-8 motor domain
ComponentsKinesin-like protein
KeywordsMOTOR PROTEIN / kinesin / microtubule / depolymerase / motility / cytoskeleton
Function / homology
Function and homology information


tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / meiotic sister chromatid segregation / mitotic spindle astral microtubule / mitotic spindle midzone / nuclear microtubule / nuclear migration along microtubule / microtubule plus-end / mitotic spindle disassembly ...tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / meiotic sister chromatid segregation / mitotic spindle astral microtubule / mitotic spindle midzone / nuclear microtubule / nuclear migration along microtubule / microtubule plus-end / mitotic spindle disassembly / plus-end-directed microtubule motor activity / microtubule depolymerization / negative regulation of microtubule polymerization / kinesin complex / microtubule-based movement / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / mitotic spindle assembly / mitotic spindle organization / microtubule binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding
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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Kinesin-like protein
Similarity search - Component
Biological speciesCandida albicans (yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.01 Å
AuthorsAllingham, J.S. / Hunter, B.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)169149 Canada
CitationJournal: Nat Commun / Year: 2022
Title: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape.
Authors: Byron Hunter / Matthieu P M H Benoit / Ana B Asenjo / Caitlin Doubleday / Daria Trofimova / Corey Frazer / Irsa Shoukat / Hernando Sosa / John S Allingham /
Abstract: Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains ...Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic. We also determined a crystal structure of microtubule-unbound CaKip3-ADP (2.0 Å) and analyzed the biochemical activity of CaKip3 and kinesin-1 mutants. These data reveal that the microtubule depolymerization activity of kinesin-8 originates from conformational changes of its motor core that are amplified by dynamic contacts between its extended loop-2 and tubulin. On curved microtubule ends, loop-1 inserts into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin and assist depolymerization. On straight tubulin protofilaments in the microtubule lattice, loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. We propose that these tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.
History
DepositionJan 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Kinesin-like protein
B: Kinesin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,5996
Polymers98,6962
Non-polymers9034
Water2,558142
1
A: Kinesin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,8003
Polymers49,3481
Non-polymers4522
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Kinesin-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,8003
Polymers49,3481
Non-polymers4522
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)43.990, 52.680, 86.750
Angle α, β, γ (deg.)86.900, 79.996, 89.924
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein Kinesin-like protein


Mass: 49348.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candida albicans (strain SC5314 / ATCC MYA-2876) (yeast)
Strain: SC5314 / ATCC MYA-2876 / Gene: KIP3, orf19.7353, CAALFM_C305720CA / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A1D8PKA4
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 142 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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 Å3/Da / Density % sol: 38.59 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.1 M MMT, 25% PEG 1500, pH 8, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08ID-1 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 14, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.01→45.91 Å / Num. obs: 49650 / % possible obs: 97.42 % / Redundancy: 2.7 % / Biso Wilson estimate: 46.62 Å2 / Rmerge(I) obs: 0.114 / Rpim(I) all: 0.082 / Rrim(I) all: 0.141 / Net I/σ(I): 4.6
Reflection shellResolution: 2.01→2.08 Å / Num. unique obs: 19655 / CC1/2: 0.149 / % possible all: 96.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0189refinement
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3LRE

3lre


Resolution: 2.01→45.91 Å / SU ML: 0.4993 / Cross valid method: FREE R-VALUE / σ(F): 1.91 / Phase error: 38.997
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2676 2472 5 %
Rwork0.2323 46999 -
obs0.2341 49471 97.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 59.75 Å2
Refinement stepCycle: LAST / Resolution: 2.01→45.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5249 0 56 142 5447
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00495403
X-RAY DIFFRACTIONf_angle_d1.04677317
X-RAY DIFFRACTIONf_chiral_restr0.0654857
X-RAY DIFFRACTIONf_plane_restr0.0059945
X-RAY DIFFRACTIONf_dihedral_angle_d25.8927755
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.01-2.050.51311280.52458X-RAY DIFFRACTION92.79
2.05-2.090.46821380.46632608X-RAY DIFFRACTION95.98
2.09-2.140.47121330.4432552X-RAY DIFFRACTION96
2.14-2.190.44851380.40782615X-RAY DIFFRACTION96.06
2.19-2.240.42231360.38712576X-RAY DIFFRACTION97.31
2.24-2.30.4051380.36922624X-RAY DIFFRACTION96.44
2.3-2.370.39491380.34772627X-RAY DIFFRACTION97.32
2.37-2.450.34281390.31212639X-RAY DIFFRACTION97.3
2.45-2.530.31791360.29222583X-RAY DIFFRACTION97.21
2.53-2.630.30991390.27242650X-RAY DIFFRACTION97.55
2.63-2.750.31321370.2572628X-RAY DIFFRACTION97.67
2.75-2.90.29421370.25272600X-RAY DIFFRACTION97.58
2.9-3.080.261390.25222640X-RAY DIFFRACTION97.99
3.08-3.320.30591390.2422638X-RAY DIFFRACTION97.92
3.32-3.650.22941390.20532641X-RAY DIFFRACTION98.16
3.65-4.180.22731380.19182617X-RAY DIFFRACTION98.22
4.18-5.270.19021400.16662665X-RAY DIFFRACTION98.25
5.27-45.910.23031400.17582638X-RAY DIFFRACTION98.13

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