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- EMDB-28139: Toxoplasma gondii apical complex (ionophore stimulated) -

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Basic information

Entry
Database: EMDB / ID: EMD-28139
TitleToxoplasma gondii apical complex (ionophore stimulated)
Map dataIonophore stimulated Toxoplasma gondii
Sample
  • Organelle or cellular component: The apical complex of Toxoplasma gondii (ionophore stimulated)
KeywordsToxoplasma gondii / apical complex / ionophore stimulated / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodelectron tomography / cryo EM
AuthorsSegev-Zarko L / Sun SY / Kim CY / Egan ES / Chiu W / Boothroyd JC
Funding support United States, 8 items
OrganizationGrant numberCountry
Other privateChan Zuckerberg Biohub Intercampus Team Award
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM121203 United States
National Institutes of Health/Office of the DirectorDP2HL137186 United States
Other privateStanford Maternal Child Health Research Institute
Other privateVaadia-BARD FI-582-2018
Other privateStanford School of Medicine Deans Postdoctoral Fellowship
CitationJournal: PNAS Nexus / Year: 2022
Title: Cryo-electron tomography with mixed-scale dense neural networks reveals key steps in deployment of invasion machinery.
Authors: Li-Av Segev-Zarko / Peter D Dahlberg / Stella Y Sun / Daniël M Pelt / Chi Yong Kim / Elizabeth S Egan / James A Sethian / Wah Chiu / John C Boothroyd /
Abstract: Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. ...Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms employs to enable repeated invasion attempts. Using the same approach, we examine merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.
History
DepositionSep 13, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28139.png

Downloads & links

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Map

FileDownload / File: emd_28139.map.gz / Format: CCP4 / Size: 1.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIonophore stimulated Toxoplasma gondii
Voxel sizeX=Y=Z: 14.16 Å
Density
Minimum - Maximum-6.50961 - 5.3333635
Average (Standard dev.)0.046263814 (±0.23100823)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin16-16166
Dimensions960928331
Spacing928960331
CellA: 13140.4795 Å / B: 13593.6 Å / C: 4686.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : The apical complex of Toxoplasma gondii (ionophore stimulated)

EntireName: The apical complex of Toxoplasma gondii (ionophore stimulated)
Components
  • Organelle or cellular component: The apical complex of Toxoplasma gondii (ionophore stimulated)

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Supramolecule #1: The apical complex of Toxoplasma gondii (ionophore stimulated)

SupramoleculeName: The apical complex of Toxoplasma gondii (ionophore stimulated)
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: RH

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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 / Component - Concentration: 1.0 x / Component - Name: Hanks balanced salt solution (HBSS)
Details: HBSS without calcium, magnesium or phenol red, supplemented with 1 mM MgCl2, 1 mM CaCl2, 10 mM NaHCO3, 20 mM HEPES, pH 7, and 1 uM calcium ionophore (A23187).
GridModel: EMS Lacey Carbon / Material: COPPER / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: LEICA EM GP
DetailsTachyzoites were released from heavily infected monolayers of HFFs by mechanical disruption of the monolayers using disposable scrapers and passage through a 25-gauge syringe into HBSS
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.15 e/Å2

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 61

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