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- EMDB-12257: Plasmodium falciparum kinesin-5 motor domain without nucleotide, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12257
TitlePlasmodium falciparum kinesin-5 motor domain without nucleotide, complexed with 14 protofilament microtubule.
Map dataAsymmetric unit of Plasmodium falciparum kinesin-5 motor domain without nucleotide, in complex with porcine 14 protofilament microtubules.
Sample
  • Complex: 14 protofilament microtubule of alpha/beta-tubulin dimers, with kinesin-5 motor domain bound 1:1 to tubulin dimers.
    • Complex: alpha/beta-tubulin
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Kinesin-5Kinesin-like protein KIF11
      • Protein or peptide: Kinesin-5Kinesin-like protein KIF11
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
Function / homology
Function and homology information


Kinesins / 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 ...Kinesins / 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 / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / plus-end-directed microtubule motor activity / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / COPI-mediated anterograde transport / kinesin complex / microtubule motor activity / 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
Kinesin-5 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Plasmodium falciparum (isolate 3D7) (eukaryote) / Pig (pig)
Methodhelical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsCook AD / Roberts A / Atherton J / Tewari R / Topf M / Moores CA
Funding support United Kingdom, 4 items
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, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateOct 27, 2021-
Current statusOct 27, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-7nb8
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12257.png

Downloads & links

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Map

FileDownload / File: emd_12257.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric unit of Plasmodium falciparum kinesin-5 motor domain without nucleotide, in complex with porcine 14 protofilament microtubules.
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum0.0 - 0.17390957
Average (Standard dev.)0.0069941035 (±0.01859888)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin116108180
Dimensions786777
Spacing677877
CellA: 93.13 Å / B: 108.42 Å / C: 107.03 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z677877
origin x/y/z0.0000.0000.000
length x/y/z93.130108.420107.030
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS108116180
NC/NR/NS677877
D min/max/mean0.0000.1740.007

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Supplemental data

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Additional map: Asymmetric (C1) reconstruction of Plasmodium falciparum kinesin-5 motor...

Fileemd_12257_additional_1.map
AnnotationAsymmetric (C1) reconstruction of Plasmodium falciparum kinesin-5 motor domain without nucleotide, in complex with porcine 14 protofilament microtubules.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Symmetry expanded reconstruction of Plasmodium falciparum kinesin-5 motor...

Fileemd_12257_additional_2.map
AnnotationSymmetry expanded reconstruction of Plasmodium falciparum kinesin-5 motor without nucleotide, in complex with porcine 14 protofilament microtubules. After motor domain 3D classification.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 14 protofilament microtubule of alpha/beta-tubulin dimers, with k...

EntireName: 14 protofilament microtubule of alpha/beta-tubulin dimers, with kinesin-5 motor domain bound 1:1 to tubulin dimers.
Components
  • Complex: 14 protofilament microtubule of alpha/beta-tubulin dimers, with kinesin-5 motor domain bound 1:1 to tubulin dimers.
    • Complex: alpha/beta-tubulin
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Kinesin-5Kinesin-like protein KIF11
      • Protein or peptide: Kinesin-5Kinesin-like protein KIF11
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

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Supramolecule #1: 14 protofilament microtubule of alpha/beta-tubulin dimers, with k...

SupramoleculeName: 14 protofilament microtubule of alpha/beta-tubulin dimers, with kinesin-5 motor domain bound 1:1 to tubulin dimers.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: alpha/beta-tubulin

SupramoleculeName: alpha/beta-tubulin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: Kinesin-5

SupramoleculeName: Kinesin-5 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Plasmodium falciparum (isolate 3D7) (eukaryote)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Organ: Brain
Molecular weightTheoretical: 50.204445 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Organ: Brain
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA

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Macromolecule #3: Kinesin-5

MacromoleculeName: Kinesin-5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7
Molecular weightTheoretical: 46.910113 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GIDPFTMLRN SYNNDKSSCV NIKVIVRCRP LNEKEKNDIN NEEVVRINNN EVILTINRNN EIYEKKYSFD YACDKDVDQK TLFNNYIYQ IVDEVLQGFN CTLFCYGQTG TGKTYTMEGK ILEHLKQYDN NKKVDLNESI NSDISYCYEL CENEDTGLIF R VTKRIFDI ...String:
GIDPFTMLRN SYNNDKSSCV NIKVIVRCRP LNEKEKNDIN NEEVVRINNN EVILTINRNN EIYEKKYSFD YACDKDVDQK TLFNNYIYQ IVDEVLQGFN CTLFCYGQTG TGKTYTMEGK ILEHLKQYDN NKKVDLNESI NSDISYCYEL CENEDTGLIF R VTKRIFDI LNKRKEEKIR HFDKNMYDFN IKISYLEIYN EELCDLLSST NENMKLRIYE DSNNKSKGLN VDKLEEKSIN SF EEIYYII CSAIKKRRTA ETAYNKKSSR SHSIFTITLI IKDINNVGES ITKIGKLNLV DLAGSENALK SSYGSLKIRQ QES CNINQS LLTLGRVINS LIENSSYIPY RDSKLTRLLQ DSLGGKTKTF IVATISPSSL CIDETLSTLD YVFRAKNIKN RPEI NIKTT

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration3.7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
20.0 mMKClpotassium chloride
2.0 mMMgCl2Magnesium chloride
2.0 mMC4H10O2S2Dithiothreitol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0003 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 296 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 335 / Average exposure time: 18.0 sec. / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3jat


Details: 3JAT was pseudosymmetrised in into different microtubule protofilament numbers using EMDB entries 5191-6. Kinesin decorated references were created using 3JAT and 3HQD.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.93 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.75 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Details: RELION 3D auto-refine / Number images used: 73684
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3hqd

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7nb8:
Plasmodium falciparum kinesin-5 motor domain without nucleotide, complexed with 14 protofilament microtubule.

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