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- EMDB-3610: Malaria-infected red blood cell section showing schizont stalled ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3610
TitleMalaria-infected red blood cell section showing schizont stalled in egress with the inhibitor E64
Map dataResin section tomogram of a Plasmodium falciparum infected erythrocyte treated with the egress inhibitor E64
Sample
  • Cell: Plasmodium falciparum infected human erythrocyte treated with the egress inhibitor E64
Biological speciesPlasmodium falciparum 3D7 (eukaryote)
Methodelectron tomography / negative staining
AuthorsHale VL / Saibil HR
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in egress.
Authors: Victoria L Hale / Jean M Watermeyer / Fiona Hackett / Gema Vizcay-Barrena / Christiaan van Ooij / James A Thomas / Matthew C Spink / Maria Harkiolaki / Elizabeth Duke / Roland A Fleck / ...Authors: Victoria L Hale / Jean M Watermeyer / Fiona Hackett / Gema Vizcay-Barrena / Christiaan van Ooij / James A Thomas / Matthew C Spink / Maria Harkiolaki / Elizabeth Duke / Roland A Fleck / Michael J Blackman / Helen R Saibil /
Abstract: In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential ...In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.
History
DepositionFeb 28, 2017-
Header (metadata) releaseMar 22, 2017-
Map releaseMar 22, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

emd_3610.png

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Map

FileDownload / File: emd_3610.map.gz / Format: CCP4 / Size: 877.9 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationResin section tomogram of a Plasmodium falciparum infected erythrocyte treated with the egress inhibitor E64
Voxel sizeX=Y=Z: 9.30825 Å
Density
Minimum - Maximum-128. - 126.
Average (Standard dev.)64.234830000000002 (±9.638585000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions19861998232
Spacing19981986232
CellA: 18597.883 Å / B: 18486.184 Å / C: 2159.5142 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z9.30824974974989.30824974823779.30825
M x/y/z19981986232
origin x/y/z0.0000.0000.000
length x/y/z18597.88318486.1842159.514
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS19981986232
D min/max/mean-128.000126.00064.235

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Supplemental data

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Sample components

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Entire : Plasmodium falciparum infected human erythrocyte treated with the...

EntireName: Plasmodium falciparum infected human erythrocyte treated with the egress inhibitor E64
Components
  • Cell: Plasmodium falciparum infected human erythrocyte treated with the egress inhibitor E64

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Supramolecule #1: Plasmodium falciparum infected human erythrocyte treated with the...

SupramoleculeName: Plasmodium falciparum infected human erythrocyte treated with the egress inhibitor E64
type: cell / ID: 1 / Parent: 0
Details: High pressure frozen and freeze-substituted cell section
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: RPMI medium
StainingType: NEGATIVE / Material: Uranyl Acetate
Sugar embeddingMaterial: HM20 / Details: Freeze substitution
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
High pressure freezingInstrument: OTHER
Details: The value given for _emd_high_pressure_freezing.instrument is Baltech HPM010. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION ...Details: The value given for _emd_high_pressure_freezing.instrument is Baltech HPM010. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
SectioningUltramicrotomy - Instrument: Leica EM UC7 / Ultramicrotomy - Temperature: 293 K / Ultramicrotomy - Final thickness: 250
Fiducial markerManufacturer: Electron microscopy sciences / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: OTHER
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 100.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 131

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