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- EMDB-3117: A Spiral Scaffold Underlies Cytoadherent Knobs in Plasmodium falc... -

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Basic information

Entry
Database: EMDB / ID: EMD-3117
TitleA Spiral Scaffold Underlies Cytoadherent Knobs in Plasmodium falciparum-Infected Erythrocytes
Map dataCryo-tomogram of uninfected erythrocyte skeleton
Sample
  • Sample: Detergent-insoluble skeleton of human erythrocyte
  • Organelle or cellular component: cytoskeleton
Keywordsmalaria / Plasmodium / knobs / cytoadherence / KAHRP / erythrocyte / skeleton / schizont / blood
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsWatermeyer JM / Hale VL / Hackett F / Clare DK / Cutts EE / Vakonakis I / Fleck RA / Blackman MJ / Saibil HR
CitationJournal: Blood / Year: 2016
Title: A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes.
Authors: Jean M Watermeyer / Victoria L Hale / Fiona Hackett / Daniel K Clare / Erin E Cutts / Ioannis Vakonakis / Roland A Fleck / Michael J Blackman / Helen R Saibil /
Abstract: Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is ...Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
History
DepositionAug 10, 2015-
Header (metadata) releaseAug 26, 2015-
Map releaseDec 23, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3117.tif

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Map

FileDownload / File: emd_3117.map.gz / Format: CCP4 / Size: 1 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-tomogram of uninfected erythrocyte skeleton
Voxel sizeX=Y=Z: 10.78 Å
Density
Minimum - Maximum128.0 - 31482.0
Average (Standard dev.)17113.0859375 (±1126.851928710000038)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0065
Dimensions19201856157
Spacing19201856157
CellA: 20007.68 Å / B: 20697.6 Å / C: 1692.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z10.7810.7810.78
M x/y/z18561920157
origin x/y/z0.0000.0000.000
length x/y/z20007.68020697.6001692.460
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0065
NC/NR/NS18561920157
D min/max/mean128.00031482.00017113.086

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Supplemental data

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Sample components

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Entire : Detergent-insoluble skeleton of human erythrocyte

EntireName: Detergent-insoluble skeleton of human erythrocyte
Components
  • Sample: Detergent-insoluble skeleton of human erythrocyte
  • Organelle or cellular component: cytoskeleton

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Supramolecule #1000: Detergent-insoluble skeleton of human erythrocyte

SupramoleculeName: Detergent-insoluble skeleton of human erythrocyte / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: cytoskeleton

SupramoleculeName: cytoskeleton / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: blood / Cell: erythrocyte / Location in cell: cytoskeleton

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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: 7.4
GridDetails: 300 mesh gold Quantifoil grid R3.5/1
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm
Specialist opticsEnergy filter - Name: Gatan Quantum
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
Detailscounting mode
DateMay 5, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 110 e/Å2
Details: Images were the average of 20 subframes recorded by the direct electron detector
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final reconstructionSoftware - Name: IMOD / Number images used: 61

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