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- EMDB-29840: Lower preconoidal ring from Toxoplasma gondii tachyzoites with Fo... -

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Basic information

Entry
Database: EMDB / ID: EMD-29840
TitleLower preconoidal ring from Toxoplasma gondii tachyzoites with Formin1 conditionally depleted
Map dataRefined subtomogram average of the lower preconoidal ring from Toxoplasma gondii tachyzoites with Formin1 conditionally depleted
Sample
  • Organelle or cellular component: Preconoidal rings
KeywordsParasite / preconoidal rings / apicomplexa / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 44.0 Å
AuthorsMartinez M / Mageswaran SK / Chang Y-W
Funding support United States, 1 items
OrganizationGrant numberCountry
David and Lucile Packard Foundation United States
CitationJournal: Nat Commun / Year: 2023
Title: Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography.
Authors: Matthew Martinez / Shrawan Kumar Mageswaran / Amandine Guérin / William David Chen / Cameron Parker Thompson / Sabine Chavin / Dominique Soldati-Favre / Boris Striepen / Yi-Wei Chang /
Abstract: The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that ...The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.
History
DepositionFeb 17, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29840.png

Downloads & links

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Map

FileDownload / File: emd_29840.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined subtomogram average of the lower preconoidal ring from Toxoplasma gondii tachyzoites with Formin1 conditionally depleted
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.65 Å/pix.
x 200 pix.
= 530. Å		2.65 Å/pix.
x 200 pix.
= 530. Å		2.65 Å/pix.
x 200 pix.
= 530. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5
Minimum - Maximum-36.532561999999999 - 30.886559999999999
Average (Standard dev.)-0.00000005238929 (±1.173127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 530.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29840_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_29840_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29840_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Preconoidal rings

EntireName: Preconoidal rings
Components
  • Organelle or cellular component: Preconoidal rings

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Supramolecule #1: Preconoidal rings

SupramoleculeName: Preconoidal rings / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: In situ structure of the lower preconoidal ring with Formin1 conditionally depleted
Source (natural)Organism: Toxoplasma gondii (eukaryote)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 99 % / Chamber temperature: 310 K / Instrument: LEICA EM GP
Details: Isolated tachyzoites were resuspended in media, and 4 uL was applied to the carbon side of the grid and front blotted for 4s.
DetailsToxoplasma gondii expressing Formin1 fused to a c-terminal mAID tag were pre-treated with 500 uM Auxin (IAA) 48 hours prior to mechanical isolation from HFF cells. Sample was averaged from within frozen-hydrated Toxoplasma gondii tachyzoites

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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
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 opticsIllumination 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 44.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo (ver. 1.1.509) / Number subtomograms used: 363
ExtractionNumber tomograms: 11 / Number images used: 366 / Method: Volumes picked interactively / Software - Name: Dynamo (ver. 1.1.509)
Details: Individual particles were picked and manually preoriented in IMOD. Particle coordinates and angles were imported into Dynamo
Final angle assignmentType: NOT APPLICABLE / Software - Name: Dynamo (ver. 1.1.509)
FSC plot (resolution estimation)

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