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Yorodumi- EMDB-12258: Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, com... -
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-Basic information
Entry | Database: EMDB / ID: EMD-12258 | |||||||||||||||
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Title | Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, complexed with 14 protofilament microtubule. | |||||||||||||||
Map data | Asymmetric unit of Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, in complex with porcine 14 protofilament microtubules. | |||||||||||||||
Sample |
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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 / 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 / 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 / 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 | |||||||||||||||
Biological species | Plasmodium falciparum (isolate NF54) (eukaryote) / Pig (pig) | |||||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 4.0 Å | |||||||||||||||
Authors | Cook AD / Roberts A / Atherton J / Tewari R / Topf M / Moores CA | |||||||||||||||
Funding support | United Kingdom, 4 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12258.map.gz | 215.4 KB | EMDB map data format | |
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Header (meta data) | emd-12258-v30.xml emd-12258.xml | 23.8 KB 23.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12258_fsc.xml | 15.3 KB | Display | FSC data file |
Images | emd_12258.png | 80.5 KB | ||
Others | emd_12258_additional_1.map.gz emd_12258_additional_2.map.gz | 170.9 MB 167.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12258 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12258 | HTTPS FTP |
-Related structure data
Related structure data | 7nbaMC 7nb8C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12258.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Asymmetric unit of Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, in complex with porcine 14 protofilament microtubules. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.39 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Symmetry expanded reconstruction of Plasmodium falciparum kinesin-5 motor...
File | emd_12258_additional_1.map | ||||||||||||
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Annotation | Symmetry expanded reconstruction of Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, in complex with porcine 14 protofilament microtubules. After motor domain 3D classification. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unsymmetrised reconstruction of Plasmodium falciparum kinesin-5 motor domain...
File | emd_12258_additional_2.map | ||||||||||||
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Annotation | Unsymmetrised reconstruction of Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP, in complex with porcine 14 protofilament microtubules. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : 14 protofilament microtubule complexed with Plasmodium falciparum...
Entire | Name: 14 protofilament microtubule complexed with Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP. Asymmetric unit. |
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Components |
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-Supramolecule #1: 14 protofilament microtubule complexed with Plasmodium falciparum...
Supramolecule | Name: 14 protofilament microtubule complexed with Plasmodium falciparum kinesin-5 motor domain bound to AMPPNP. Asymmetric unit. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Plasmodium falciparum (isolate NF54) (eukaryote) |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
-Macromolecule #1: Tubulin alpha-1B chain
Macromolecule | Name: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Pig (pig) / Organ: BRAIN |
Molecular weight | Theoretical: 50.204445 KDa |
Sequence | String: 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 |
-Macromolecule #2: Tubulin beta chain
Macromolecule | Name: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Pig (pig) / Organ: Brain |
Molecular weight | Theoretical: 49.90777 KDa |
Sequence | String: 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 |
-Macromolecule #3: Kinesin motor domain-containing protein,Kinesin motor domain-cont...
Macromolecule | Name: Kinesin motor domain-containing protein,Kinesin motor domain-containing protein type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Plasmodium falciparum (isolate NF54) (eukaryote) / Strain: isolate NF54 |
Molecular weight | Theoretical: 46.910113 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: 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 |
-Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP |
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Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ChemComp-GTP: |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
Macromolecule | Name: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: G2P |
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Molecular weight | Theoretical: 521.208 Da |
Chemical component information | ChemComp-G2P: |
-Macromolecule #7: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 3.9 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: C-flat-2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 300.0 kPa | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 296 K / Instrument: FEI VITROBOT MARK IV 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.. |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1320 / Average exposure time: 18.0 sec. / Average electron dose: 58.0 e/Å2 |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |