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- EMDB-42118: Toxoplasma gondii apical end with ICMAP1 conditionally knocked down -

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Basic information

Entry
Database: EMDB / ID: EMD-42118
TitleToxoplasma gondii apical end with ICMAP1 conditionally knocked down
Map dataToxoplasma gondii apical end with ICMAP1 conditionally knocked down
Sample
  • Cell: Toxoplasma Gondii tachyzoite apical complex with ICMAP1 conditionally knocked down
KeywordsParasite / Conoid / Microtubules / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodelectron tomography / cryo EM
AuthorsMartinez M / Dos Santos Pacheco N / Chang Y-W / Soldati-Favre D
Funding support United States, Switzerland, European Union, 4 items
OrganizationGrant numberCountry
David and Lucile Packard Foundation United States
Swiss National Science Foundation310030_185325 Switzerland
European Research Council (ERC)695596European Union
Burroughs Wellcome Fund1022785 United States
CitationJournal: Nat Commun / Year: 2024
Title: Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins.
Authors: Nicolas Dos Santos Pacheco / Albert Tell I Puig / Amandine Guérin / Matthew Martinez / Bohumil Maco / Nicolò Tosetti / Estefanía Delgado-Betancourt / Matteo Lunghi / Boris Striepen / Yi- ...Authors: Nicolas Dos Santos Pacheco / Albert Tell I Puig / Amandine Guérin / Matthew Martinez / Bohumil Maco / Nicolò Tosetti / Estefanía Delgado-Betancourt / Matteo Lunghi / Boris Striepen / Yi-Wei Chang / Dominique Soldati-Favre /
Abstract: In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with ...In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.
History
DepositionSep 26, 2023-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42118.png

Downloads & links

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Map

FileDownload / File: emd_42118.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationToxoplasma gondii apical end with ICMAP1 conditionally knocked down
Voxel sizeX=Y=Z: 10.6 Å
Density
Minimum - Maximum-1218.0 - 372.0
Average (Standard dev.)56.713875000000002 (±32.042476999999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-209209-401
Dimensions14401023800
Spacing10231440800
CellA: 10843.801 Å / B: 15264.001 Å / C: 8480.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Toxoplasma Gondii tachyzoite apical complex with ICMAP1 condition...

EntireName: Toxoplasma Gondii tachyzoite apical complex with ICMAP1 conditionally knocked down
Components
  • Cell: Toxoplasma Gondii tachyzoite apical complex with ICMAP1 conditionally knocked down

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Supramolecule #1: Toxoplasma Gondii tachyzoite apical complex with ICMAP1 condition...

SupramoleculeName: Toxoplasma Gondii tachyzoite apical complex with ICMAP1 conditionally knocked down
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Toxoplasma gondii (eukaryote)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Details: DMEM supplemented with 5% fetal bovine serum and 2 mM glutamine
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 310 K / Instrument: LEICA EM GP / Details: Front blot for 4 seconds before plunging.
DetailsEgressed tachyzoites from a culture of confluent HFF cells. ICMAP1 knockdown induced by treatment with 50 nM rapamycin for 48 hours prior to egress.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Ted Pella, Inc / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
SoftwareName: SerialEM (ver. 3.8)
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.4 sec. / Average electron dose: 2.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsRaw frames were gain normalized in serialEM and motion corrected using the alignframes command in IMOD
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.11.5) / Number images used: 61

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