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- PDB-7nba: Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, com... -

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Basic information

Entry
Database: PDB / ID: 7nba
TitlePlasmodium falciparum kinesin-5 motor domain bound to AMPPNP, complexed with 14 protofilament microtubule.
Components
  • Kinesin motor domain-containing protein,Kinesin motor domain-containing protein
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN / Cytoskeleton / mitotic
Function / homology
Function and homology information


microtubule motor activity => GO:0003777 / 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 ...microtubule motor activity => GO:0003777 / 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 / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / COPI-mediated anterograde transport / microtubule-based movement / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site ...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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin motor domain-containing protein
Similarity search - Component
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 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

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
K: Kinesin motor domain-containing protein,Kinesin motor domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,6469
Polymers147,0223
Non-polymers1,6236
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9170 Å2
ΔGint-62 kcal/mol
Surface area45970 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 motor domain-containing protein,Kinesin motor domain-containing protein


Mass: 46910.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate NF54) (eukaryote)
Strain: isolate NF54 / Gene: PFNF54_00523 / Plasmid: pET151 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: W7K044

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Non-polymers , 4 types, 6 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.: 3 / 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
#7: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, 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

ComponentName: 14 protofilament microtubule complexed with Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP. Asymmetric unit.
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Plasmodium falciparum (isolate NF54) (eukaryote)
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
55 mMAdenylyl-imidodiphosphateC10H17N6O12P31
SpecimenConc.: 3.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 296 K
Details: 5 uM microtubules were applied to the grid and incubated for 30 seconds. The grid was blotted, then 40 uM kinesin motor domain added, incubated for 30 seconds, then blotted. 40 uM Kinesin ...Details: 5 uM microtubules were applied to the grid and incubated for 30 seconds. The grid was blotted, then 40 uM kinesin motor domain added, incubated for 30 seconds, then blotted. 40 uM Kinesin motor domain was again added, incubated for 40 seconds, blotted, then 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: 1320

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Flex-EMmodel fitting
9Rosettamodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -25.75 ° / Axial rise/subunit: 9 Å / Axial symmetry: C1
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166375 / Details: RELION 3D auto-refine / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building
IDPDB-ID 3D fitting-ID
13HQD

3hqd

1
23JAT

3jat

1

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