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- PDB-7nb8: Plasmodium falciparum kinesin-5 motor domain without nucleotide, ... -

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Basic information

Entry
Database: PDB / ID: 7nb8
TitlePlasmodium falciparum kinesin-5 motor domain without nucleotide, complexed with 14 protofilament microtubule.
Components
  • Kinesin-5
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN / Cytoskeleton / mitosis
Function / homology
Function and homology information


Kinesins / spindle elongation / plus-end-directed microtubule motor activity / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase ...Kinesins / spindle elongation / plus-end-directed microtubule motor activity / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / kinesin complex / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / microtubule motor activity / COPI-mediated anterograde transport / microtubule-based movement / mitotic spindle assembly / spindle microtubule / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / GTPase activity / GTP binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
: / 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 / Tubulin-beta mRNA autoregulation signal. ...: / 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 / Tubulin-beta mRNA autoregulation signal. / 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 / Kinesin-5 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsCook, A.D. / Roberts, A. / Atherton, J. / Tewari, R. / Topf, M. / Moores, C.A.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/J003867/1 United Kingdom
Wellcome Trust101311-10 United Kingdom
Wellcome Trust079605/Z/06/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/L014211/1 United Kingdom
CitationJournal: J Biol Chem / Year: 2021
Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition.
Authors: Alexander D Cook / Anthony J Roberts / Joseph Atherton / Rita Tewari / Maya Topf / Carolyn A Moores /
Abstract: Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the ...Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.
History
DepositionJan 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 27, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc / Item: _citation.journal_volume / _em_admin.last_update
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)148,1157
Polymers147,0223
Non-polymers1,0934
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8270 Å2
ΔGint-48 kcal/mol
Surface area45190 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABK

#1: 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) / Organ: Brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Brain / References: UniProt: P02554
#3: Protein Kinesin-5


Mass: 46910.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7 / Gene: PF3D7_0317500 / Plasmid: pET151 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: O77382

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Non-polymers , 3 types, 4 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#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

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Details

Has ligand of interestN

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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
IDNameTypeEntity IDParent-IDSource
114 protofilament microtubule of alpha/beta-tubulin dimers, with kinesin-5 motor domain bound 1:1 to tubulin dimers.COMPLEX#1-#30MULTIPLE SOURCES
2alpha/beta-tubulinCOMPLEX#1-#21NATURAL
3Kinesin-5COMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa (pig)9823
23Plasmodium falciparum (isolate 3D7) (eukaryote)36329
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
220 mMpotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
42 mMDithiothreitolC4H10O2S21
SpecimenConc.: 3.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 296 K
Details: 3.5 uM microtubules were incubated to the grid and incubated for 30 seconds. The grid was blotted, then 40 uM kinesin motor domain added and incubated for 30 seconds. The grid was again ...Details: 3.5 uM microtubules were incubated to the grid and incubated for 30 seconds. The grid was blotted, then 40 uM kinesin motor domain added and incubated for 30 seconds. The grid was again blotted and 40 uM kinesin motor domain added for 30 seconds. Then the grid was blotted and plunge frozen.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2 mm
Image recordingAverage exposure time: 18 sec. / Electron dose: 58 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 335

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
7Flex-EMmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -25.75 ° / Axial rise/subunit: 8.93 Å / Axial symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73684 / Details: RELION 3D auto-refine / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 3HQD

3hqd


Accession code: 3HQD / Source name: PDB / Type: experimental model

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