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- EMDB-28140: Toxoplasma gondii apical complex (non-stimulated) -

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Basic information

Entry
Database: EMDB / ID: EMD-28140
TitleToxoplasma gondii apical complex (non-stimulated)
Map dataToxoplasma gondii apical complex (non-stimulated)
Sample
  • Organelle or cellular component: The apical complex of Toxoplasma gondii (non-stimulated)
KeywordsToxoplasma gondii / apical complex / non-stimulated / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodelectron tomography / cryo EM
AuthorsSegev-Zarko L / Sun SY / Chiu W / Boothroyd JC
Funding support United States, 2 items
OrganizationGrant numberCountry
Other private United States
Other government
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_28140.png

Downloads & links

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Map

FileDownload / File: emd_28140.map.gz / Format: CCP4 / Size: 355.1 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationToxoplasma gondii apical complex (non-stimulated)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.84 Å/pix.
x 209 pix.
= 2892.56 Å		13.84 Å/pix.
x 928 pix.
= 12843.521 Å		13.84 Å/pix.
x 960 pix.
= 13286.4 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 13.84 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-584.0 - 482.0
Average (Standard dev.)1.8708776 (±18.162770999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00105
Dimensions928960209
Spacing960928209
CellA: 13286.4 Å / B: 12843.5205 Å / C: 2892.56 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 (non-stimulated)

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

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

SupramoleculeName: The apical complex of Toxoplasma gondii (non-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: 7.2 / Component - Concentration: 1.0 x / Component - Name: Endo buffer
Details: 45 mM potassium sulfate, 106 mM sucrose, 10 mM magnesium sulfate, 20 mM Tris buffer pH 7.2, 5 mM glucose and 0.35% bovine serum albumin
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: 297 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 HPEB
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 26000
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

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

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