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- EMDB-3116: A Spiral Scaffold Underlies Cytoadherent Knobs in Plasmodium falc... -

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Entry
Database: EMDB / ID: EMD-3116
TitleA Spiral Scaffold Underlies Cytoadherent Knobs in Plasmodium falciparum-Infected Erythrocytes
Map dataCryo-tomogram of detergent-extracted cytoskeleton of P. falciparum-infected erythrocyte, schiztont stage, showing knob skeletons. Map has been NAD and high-pass filtered to remove background gradient.
Sample
  • Sample: Detergent-resistant skeleton of P. falciparum schizont
  • Organelle or cellular component: Cytoskeleton with knobs
Keywordsmalaria / Plasmodium / knobs / cytoadherence / KAHRP / erythrocyte / skeleton / schizont / blood
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
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

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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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3116.tif

Downloads & links

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Map

FileDownload / File: emd_3116.map.gz / Format: CCP4 / Size: 770 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-tomogram of detergent-extracted cytoskeleton of P. falciparum-infected erythrocyte, schiztont stage, showing knob skeletons. Map has been NAD and high-pass filtered to remove background gradient.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.78 Å/pix.
x 116 pix.
= 1250.48 Å		10.78 Å/pix.
x 1920 pix.
= 20697.6 Å		10.78 Å/pix.
x 1856 pix.
= 20007.68 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.78 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum0.0 - 32510.0
Average (Standard dev.)16730.369140629998583 (±1202.041992189999974)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0057
Dimensions19201856116
Spacing19201856116
CellA: 20007.68 Å / B: 20697.6 Å / C: 1250.48 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/z18561920116
origin x/y/z0.0000.0000.000
length x/y/z20007.68020697.6001250.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0057
NC/NR/NS18561920116
D min/max/mean0.00032510.00016730.369

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Supplemental data

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Sample components

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Entire : Detergent-resistant skeleton of P. falciparum schizont

EntireName: Detergent-resistant skeleton of P. falciparum schizont
Components
  • Sample: Detergent-resistant skeleton of P. falciparum schizont
  • Organelle or cellular component: Cytoskeleton with knobs

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Supramolecule #1000: Detergent-resistant skeleton of P. falciparum schizont

SupramoleculeName: Detergent-resistant skeleton of P. falciparum schizont
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Cytoskeleton with knobs

SupramoleculeName: Cytoskeleton with knobs / type: organelle_or_cellular_component / ID: 1
Details: Mature schizonts were lysed and detergent-extracted on EM grids, then plunge-frozen.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Plasmodium falciparum (malaria parasite P. falciparum)
Strain: 3D7 / synonym: malaria parasite / Cell: schizont / Location in cell: erythrocyte 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 copper grid with lacy carbon support and thin carbon overlay
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: Gatan Quantum
Detailscounting mode
DateAug 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 53 e/Å2
Details: Images were the average of 6 subframes recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -69 ° / Tilt series - Axis1 - Max angle: 53 ° / Tilt series - Axis1 - Angle increment: 2 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 62
CTF correctionDetails: each image

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