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- PDB-7kiy: Plasmodium falciparum RhopH complex in soluble form -

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Basic information

Entry
Database: PDB / ID: 7kiy
TitlePlasmodium falciparum RhopH complex in soluble form
Components
  • Cytoadherence linked asexual protein 3
  • High molecular weight rhoptry protein 3
  • High molecular weight rhoptry protein-2
KeywordsMEMBRANE PROTEIN / Plasmodium falciparum / malaria / RhopH complex
Function / homologyUncharacterized protein / High molecular weight rhoptry protein-2
Function and homology information
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsSchureck, M.A. / Darling, J.E. / Merk, A. / Subramaniam, S. / Desai, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Elife / Year: 2021
Title: Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake.
Authors: Marc A Schureck / Joseph E Darling / Alan Merk / Jinfeng Shao / Geervani Daggupati / Prakash Srinivasan / Paul Dominic B Olinares / Michael P Rout / Brian T Chait / Kurt Wollenberg / Sriram ...Authors: Marc A Schureck / Joseph E Darling / Alan Merk / Jinfeng Shao / Geervani Daggupati / Prakash Srinivasan / Paul Dominic B Olinares / Michael P Rout / Brian T Chait / Kurt Wollenberg / Sriram Subramaniam / Sanjay A Desai /
Abstract: Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through ...Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through subcellular sites to serve these essential functions is unknown. We show that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. We present a 2.9 Å single-particle cryo-electron microscopy structure of the trafficking complex, revealing that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. We propose a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms.
History
DepositionOct 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Cytoadherence linked asexual protein 3
B: High molecular weight rhoptry protein-2
C: High molecular weight rhoptry protein 3


Theoretical massNumber of molelcules
Total (without water)444,9283
Polymers444,9283
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cytoadherence linked asexual protein 3


Mass: 177069.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (malaria parasite P. falciparum)
#2: Protein High molecular weight rhoptry protein-2 / RhopH2


Mass: 162877.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (malaria parasite P. falciparum)
References: UniProt: Q8I060
#3: Protein High molecular weight rhoptry protein 3 / RhopH3


Mass: 104981.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (malaria parasite P. falciparum)
References: UniProt: A0A024X9S2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RhopH complex / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.434 MDa / Experimental value: YES
Source (natural)Organism: Plasmodium falciparum (malaria parasite P. falciparum)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMtris(hydroxymethyl)aminomethaneTris1
2200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 0.4 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1310
Image scansMovie frames/image: 58 / Used frames/image: 1-58

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
7Coot0.8.9.1model fitting
9PHENIX1.16-3549model refinement
Image processingDetails: All cryo-EM image processing was performed in RELION 3.0.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 311390
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68216 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 97.81 / Protocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 97.81 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007520340
ELECTRON MICROSCOPYf_angle_d0.925827421
ELECTRON MICROSCOPYf_chiral_restr0.05432973
ELECTRON MICROSCOPYf_plane_restr0.0053444
ELECTRON MICROSCOPYf_dihedral_angle_d14.270712260

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