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- EMDB-26672: Rhoptry secretory apparatus of Plasmodium falciparum with an asso... -

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Basic information

Entry
Database: EMDB / ID: EMD-26672
TitleRhoptry secretory apparatus of Plasmodium falciparum with an associated rhoptry tip from a fused rhoptry pair
Map dataRhoptry secretory apparatus of Plasmodium falciparum with an associated rhoptry tip from a fused rhoptry pair
Sample
  • Cell: Plasmodium falciparum merozoite
    • Complex: Rhoptry secretory apparatus
    • Organelle or cellular component: Rhoptry
KeywordsExocytosis Membrane Parasite / CELL INVASION
Biological speciesPlasmodium falciparum 3D7 (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 50.0 Å
AuthorsMartinez M / Chang Y-W
Funding support United States, 1 items
OrganizationGrant numberCountry
David and Lucile Packard Foundation United States
CitationJournal: Nat Microbiol / Year: 2022
Title: Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography.
Authors: Matthew Martinez / William David Chen / Marta Mendonça Cova / Petra Molnár / Shrawan Kumar Mageswaran / Amandine Guérin / Audrey R Odom John / Maryse Lebrun / Yi-Wei Chang /
Abstract: Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory ...Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is unknown whether a similar molecular machinery exists in the malaria parasite Plasmodium. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct apical vesicle docking the tips of the two rhoptries to the RSA. We also discover two additional rhoptry organizations that lack the apical vesicle. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to different rhoptry organizations. Our results highlight previously unknown steps in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.
History
DepositionApr 15, 2022-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26672.png

Downloads & links

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Map

FileDownload / File: emd_26672.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRhoptry secretory apparatus of Plasmodium falciparum with an associated rhoptry tip from a fused rhoptry pair
Voxel sizeX=Y=Z: 10.6 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-11.248218 - 12.412521
Average (Standard dev.)0.0000026538114 (±1.6596062)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-45-45-45
Dimensions909090
Spacing909090
CellA=B=C: 954.00006 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26672_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Rhoptry secretory apparatus of Plasmodium falciparum with an...

Fileemd_26672_half_map_1.map
AnnotationRhoptry secretory apparatus of Plasmodium falciparum with an associated rhoptry tip from a fused rhoptry pair
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Rhoptry secretory apparatus of Plasmodium falciparum with an...

Fileemd_26672_half_map_2.map
AnnotationRhoptry secretory apparatus of Plasmodium falciparum with an associated rhoptry tip from a fused rhoptry pair
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Plasmodium falciparum merozoite

EntireName: Plasmodium falciparum merozoite
Components
  • Cell: Plasmodium falciparum merozoite
    • Complex: Rhoptry secretory apparatus
    • Organelle or cellular component: Rhoptry

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Supramolecule #1: Plasmodium falciparum merozoite

SupramoleculeName: Plasmodium falciparum merozoite / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)

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Supramolecule #2: Rhoptry secretory apparatus

SupramoleculeName: Rhoptry secretory apparatus / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: 8-fold symmetric protein structure, anchored into the plasma membrane, which docks a fused rhoptry tip to the site of exocytosis at the plasma membrane
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote) / Organelle: Apical complex / Location in cell: Cytoplasm

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Supramolecule #3: Rhoptry

SupramoleculeName: Rhoptry / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: The rhoptry tip from a fused rhoptry pair docked at the rhoptry secretory apparatus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Details: RPMI medium modified with 27 mM NaHCO3, 11 mM glucose, 5 mM HEPES, 0.01 mM thymidine, 1 mM sodium pyruvate, 0.37 mM hypoxanthine, 10 ug/mL gentamicin, and 5 g/L Albumax
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: 95 % / Chamber temperature: 297 K / Instrument: LEICA EM GP
Details: Blot for 4 or 6 seconds from the front before plunging.
DetailsMechanically isolated merozoites from a highly synchronous culture of Plasmodium falciparum schizonts

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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) / Number grids imaged: 9 / Average exposure time: 0.45 sec. / Average electron dose: 2.5 e/Å2
Details: Images were collected in dose-fractionation mode with 0.1 second exposure per frame, for a total of 0.4 or 0.5 seconds per tilt image
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: 1.0 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

Final reconstructionApplied symmetry - Point group: C8 (8 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: FSC 0.143 CUT-OFF
Details: FSC was calculated using the "adaptive bandpass filter" function in Dynamo. FSC was calculated between the two half maps using a mask that covered the rhoptry secretory apparatus structure ...Details: FSC was calculated using the "adaptive bandpass filter" function in Dynamo. FSC was calculated between the two half maps using a mask that covered the rhoptry secretory apparatus structure and the top of the associated rhoptry tip.
Number subtomograms used: 36
ExtractionNumber tomograms: 36 / Number images used: 36
Reference model: Average of manually preoriented particles with no search refinement
Method: Volumes picked manually
Software:
Namedetails
IMOD (ver. 4.11.5)Volume selection
Dynamo (ver. 1.1.509)Volume extraction

Details: In IMOD, volumes were manually selected and preoriented
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Dynamo (ver. 1.1.509)
FSC plot (resolution estimation)

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