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- EMDB-26006: 3D reconstruction of detergent-extract Toxoplasma gondii cells at... -

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Entry
Database: EMDB / ID: EMD-26006
Title3D reconstruction of detergent-extract Toxoplasma gondii cells at the apical end
Map data
Sample
  • Organelle or cellular component: Detergent extract Toxoplasma gondii cells at the apical end containing conoid and microtubule elements
Keywordsparasites / Toxoplasma gondii / cytoskeleton / conoid / microtubules / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodelectron tomography / cryo EM
AuthorsSun SY / Segev-Zarko L
Funding support United States, 5 items
OrganizationGrant numberCountry
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)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM121203 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R21MH125285-01A1 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-ET of parasites gives subnanometer insight into tubulin-based structures.
Authors: Stella Y Sun / Li-Av Segev-Zarko / Muyuan Chen / Grigore D Pintilie / Michael F Schmid / Steven J Ludtke / John C Boothroyd / Wah Chiu /
Abstract: Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in , an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing ...Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in , an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.
History
DepositionJan 21, 2022-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_26006.png

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Map

FileDownload / File: emd_26006.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 11.074 Å
Density
Minimum - Maximum-39.521749999999997 - 27.713785000000001
Average (Standard dev.)0.000000000092836 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-512-512-150
Dimensions10241024300
Spacing10241024300
CellA: 11339.776 Å / B: 11339.776 Å / C: 3322.2002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z11.07411.07411.074
M x/y/z10241024300
origin x/y/z0.0000.0000.000
length x/y/z11339.77611339.7763322.200
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS-512-512-150
NC/NR/NS10241024300
D min/max/mean-39.52227.7140.000

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Supplemental data

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Sample components

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Entire : Detergent extract Toxoplasma gondii cells at the apical end conta...

EntireName: Detergent extract Toxoplasma gondii cells at the apical end containing conoid and microtubule elements
Components
  • Organelle or cellular component: Detergent extract Toxoplasma gondii cells at the apical end containing conoid and microtubule elements

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Supramolecule #1: Detergent extract Toxoplasma gondii cells at the apical end conta...

SupramoleculeName: Detergent extract Toxoplasma gondii cells at the apical end containing conoid and microtubule elements
type: organelle_or_cellular_component / 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 statefilament

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Sample preparation

BufferpH: 7.2 / Details: Phosphate-Buffered Saline
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: LEICA EM GP
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 53000
Specialist opticsEnergy 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) / Detector mode: COUNTING / Average electron dose: 90.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: EMAN2 / Number images used: 41

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