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- EMDB-12600: Structure of the Toxoplasma gondii kinase Ron13, kinase-dead mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-12600
TitleStructure of the Toxoplasma gondii kinase Ron13, kinase-dead mutant
Map dataRon13 cryo-EM map
Sample
  • Organelle or cellular component: Secretory pathway kinase Ron13
    • Protein or peptide: Protein kinase domain-containing protein
Function / homology
Function and homology information


membrane => GO:0016020 / protein kinase activity / ATP binding
Similarity search - Function
Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Protein kinase domain-containing protein
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote) / Toxoplasma gondii (strain ATCC 50853 / GT1) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.125 Å
AuthorsKorkhov VM / Mehta V
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_166678 Switzerland
Swiss National Science Foundation310030_185325 Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Structural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion.
Authors: Gaëlle Lentini / Rouaa Ben Chaabene / Oscar Vadas / Chandra Ramakrishnan / Budhaditya Mukherjee / Ved Mehta / Matteo Lunghi / Jonas Grossmann / Bohumil Maco / Rémy Visentin / Adrian B Hehl ...Authors: Gaëlle Lentini / Rouaa Ben Chaabene / Oscar Vadas / Chandra Ramakrishnan / Budhaditya Mukherjee / Ved Mehta / Matteo Lunghi / Jonas Grossmann / Bohumil Maco / Rémy Visentin / Adrian B Hehl / Volodymyr M Korkhov / Dominique Soldati-Favre /
Abstract: Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. ...Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.
History
DepositionMar 13, 2021-
Header (metadata) releaseMay 26, 2021-
Map releaseMay 26, 2021-
UpdateJul 7, 2021-
Current statusJul 7, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nur
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12600.png

Downloads & links

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Map

FileDownload / File: emd_12600.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRon13 cryo-EM map
Voxel sizeX=Y=Z: 0.8544 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.08203783 - 0.13599563
Average (Standard dev.)0.00011052869 (±0.002803429)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 256.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.85440.85440.8544
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z256.320256.320256.320
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0820.1360.000

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Supplemental data

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Sample components

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Entire : Secretory pathway kinase Ron13

EntireName: Secretory pathway kinase Ron13
Components
  • Organelle or cellular component: Secretory pathway kinase Ron13
    • Protein or peptide: Protein kinase domain-containing protein

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Supramolecule #1: Secretory pathway kinase Ron13

SupramoleculeName: Secretory pathway kinase Ron13 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Toxoplasma gondii (eukaryote)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac

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Macromolecule #1: Protein kinase domain-containing protein

MacromoleculeName: Protein kinase domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (strain ATCC 50853 / GT1) (eukaryote)
Strain: ATCC 50853 / GT1
Molecular weightTheoretical: 126.41968 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GAGTTGGKKD DQALSFLGDT KSASELNPPR LVCPDKPPTL PPREEQVRKA YALPLCELPW DDLGPMLGSG TFGRVYPLRR PACTEVTKG FVGRKFAVKI FWLKRKGMMN LFDTISQGGT PSAEQTDPGT IAAIKSEIRS LPTSSSAFRD MVRIADPTVD V EKIKGMAD ...String:
GAGTTGGKKD DQALSFLGDT KSASELNPPR LVCPDKPPTL PPREEQVRKA YALPLCELPW DDLGPMLGSG TFGRVYPLRR PACTEVTKG FVGRKFAVKI FWLKRKGMMN LFDTISQGGT PSAEQTDPGT IAAIKSEIRS LPTSSSAFRD MVRIADPTVD V EKIKGMAD SLTVETIMKE AKTLRTVINT NGFYTEVGET GTIFTQMEKF VQAHRPEIWS TLSKASQEAQ ASKYAEIGLA DN HWSLPLA RVLVKDKNDV KHWALLIELF DGDLQPKTDK TGYSLDGWNA KSGGNVVLRE IFSSREALIG LTSKLVKPFV VMQ NLYSLG HFAIKPPNLL YKYFPGEKGR ASRLSVAAGD FGMAGLLHGD MILRGTLAFM APEMERVSGG LVAKPSYDVY ALAL TLASF WTAATELRDH YPWVEKCIKP TLKKMKDAPE FTFLRFASKT GPKLYEADTI YALSTCFAVG GKVEKLYHTG MPLLI RLKL SQMADPEPLA RVSMRHARFV FKAYAMLDKL LRAPQSEANA ETREEQLKQL QSLHIVQFLL FYLRMEPLTA ARDNTQ SYR RLARALLDFA RLDPVYQAAT ETVQPLPYEF FTEQKDWQNV KVEVSGSEVD ETIRKLRTSL TRDRSLSEDS WADLVDI MF GVSLDGLREV VTRVVYSRKT FLLEEKIGNA VKEAVAATYK FDPNTQLIAE DAPDRLFEVV RTDLGLSYPD DSELGRFL V HRVSKSHTAW ATVDRLARQA LRLALRREER TRQVYEQLLS GEKPSSESEK AFFDSVFSAV SVVSEANYFG LFWDFPSAG LFGVPPEEMQ AYVRKTHLAF VGKMWPVETQ KKILEAAVRV TVRGLNASLP ASLVDVYATV FAALPTKAPV SPPFLYGLER EEYSSLLFD AKLPEFKEMV AFWATRHELN IAVQTAVGKI PDATNLSDED IEKQLEGMLP AHLRSPSPAR FGWPPEAVAD N IRLFIREA KDELALHGPD MVHNRIRVNG RSKPPRRAAF LFHEIFRKAI AFKKDISVLQ FNQFFTDILK QSFDPQCRRF IA EVKKRVK SAPAEYVRVA DTEAVAPLFE GEGKDILKLV AVDPAARASD PEPNNCFLWT QAFLDDKTIV VSTGSSGSSG HHH HHHHHH H

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration15 mg/mL
BufferpH: 7.5
Details: 5 mM Tris pH 7.5 / 200 mM NaCl / 0.5 mM EDTA / 3 mM DTT
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.125 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 320723
FSC plot (resolution estimation)

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