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- PDB-6mpv: Cryo-electron microscopy structure of Plasmodium falciparum Rh5/C... -

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Basic information

Entry
Database: PDB / ID: 6mpv
TitleCryo-electron microscopy structure of Plasmodium falciparum Rh5/CyRPA/Ripr invasion complex
Components
  • (PfRipr) x 2
  • Cysteine-rich protective antigen
  • Reticulocyte binding protein 5
KeywordsCELL INVASION / Invasion Ligand Protein complex
Function / homology
Function and homology information


microneme lumen / microneme / symbiont entry into host / apical part of cell / cytoplasmic vesicle / host extracellular space / host cell plasma membrane / protein-containing complex / extracellular region / plasma membrane
Similarity search - Function
Rh5 coiled-coil domain / Rh5 coiled-coil domain / Cysteine-rich protective antigen 6 bladed domain / Cysteine-Rich Protective Antigen 6 bladed domain
Similarity search - Domain/homology
Reticulocyte binding protein 5 / Cysteine-rich protective antigen
Similarity search - Component
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
Plasmodium falciparum 3D7 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.17 Å
AuthorsWilson, W. / Zhiheng, Y. / Cowman, A.F.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)GNT637406 Australia
CitationJournal: Nature / Year: 2019
Title: Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.
Authors: Wilson Wong / Rick Huang / Sebastien Menant / Chuan Hong / Jarrod J Sandow / Richard W Birkinshaw / Julie Healer / Anthony N Hodder / Usheer Kanjee / Christopher J Tonkin / Denise Heckmann / ...Authors: Wilson Wong / Rick Huang / Sebastien Menant / Chuan Hong / Jarrod J Sandow / Richard W Birkinshaw / Julie Healer / Anthony N Hodder / Usheer Kanjee / Christopher J Tonkin / Denise Heckmann / Vladislav Soroka / Teit Max Moscote Søgaard / Thomas Jørgensen / Manoj T Duraisingh / Peter E Czabotar / Willem A de Jongh / Wai-Hong Tham / Andrew I Webb / Zhiheng Yu / Alan F Cowman /
Abstract: Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions ...Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.
History
DepositionOct 8, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 26, 2018Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 16, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Cysteine-rich protective antigen
B: Reticulocyte binding protein 5
C: PfRipr
D: PfRipr


Theoretical massNumber of molelcules
Total (without water)80,8974
Polymers80,8974
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cysteine-rich protective antigen


Mass: 39270.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7 / Gene: PF3D7_0423800
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q8IFM8
#2: Protein Reticulocyte binding protein 5


Mass: 39803.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (malaria parasite P. falciparum)
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: B2L3N7
#3: Protein/peptide PfRipr


Mass: 1379.692 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum 3D7 (eukaryote) / Production host: Drosophila nebulosa (fry)
#4: Protein/peptide PfRipr


Mass: 443.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum 3D7 (eukaryote) / Production host: Drosophila nebulosa (fry)
Sequence detailsThe sample sequence corresponding to PfRipr is the following: ...The sample sequence corresponding to PfRipr is the following: IDLIEGIFYEKNEIDKLTFSLDHRVRDNLKTDLILNNNGENDYAYLNKYVYTILNRDSTEKIKTFFSHNKDMKSCDYFISKEYNSSDKTNQICYKKTFCGVVIPNSEEIKTNKITNDKLYCAHFNSTHIIIYYISQPLLLEPHVVYEETFFEKGKNDQINCQGMYISLRSVHVHTHNAILQQETLTYIKNLCDGKNNCKFDFDSIKYENKSLTHYLFFINIQYQCISPLNLQENEMCDVYNDDTHKATCKYGFNKIELLKNVCEENYRCTQDICSVNQFCDGENETCTCKTSLLPSAKNNCEYNDLCTVLNCPENSTCEQIGNGKKAECKCENGKYYHNNKCYTKNDLELAIKIEPHKKEKF3YKNNLYQGKALKPEYIFMQCENGFSIEVINAYVSCYRVSFNLNKLKYVTESLKKMCDGKTKCAYGNTIDPIDDLNHHNICNNFNTIFKYDYLCVFNNQNITSDKNSHLHSNIPSLYNSSILPDINKSKFHLISRNSRTNQYPHNNISMLEIQNEISSHNSNQFSTDPHTNSNNINNMNIKKVEIFRSRFSSKLQCQGGKINIDKAILKGGEGCNDLLLTNSLKSYCNDLSECDIGLIYHFDTYCINDQYLFVSYSCSNLCNKCHNNSTCYGNRFNYDCFCDNPYISKYGNKLCERPNDCESVLCSQNQVCQILPNDKLICQCEEGYKNVKGKCVPDNKCDLSCPSNKVCVIENGKQTCKCSERFVLENGVCICANDYKMEDGINCIAKNKCKRKEYENICTNPNEMCAYNEETDIVKCECKEHYYRSSRGECILNDYCKDINCKENEECSIVNFKPECVCKENLKKNNKGECIYENSCLINEGNCPKDSKCIYREYKPHECVCNKQGHVAVNGKCVLEDKCVHNKKCSENSICVNVMNKEPICVCTYNYYKKDGVCLIQNPCLKDNGGCSRNSECTFKYSKINCTCKENYKNKDDSCVPNTNEYDESFTFQYNDDASIILGACGMIEFSYIYNQIIWKINNSKESYVFYYDYPTAGNIEVQIKNEIFHTIIYLKKKIGNSVIYDDFQVDHQTCIYENVFYYSNQNSAWSHPQFEK

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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: PfRh5/CyRPA/PfRipr complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 8.5 / Details: 20 mM Tris, pH 8.5, 150 mM NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 / Nominal magnification: 105000 X / Calibrated magnification: 48077 X / Cs: 0.01 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 92.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 12974
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Spherical aberration corrector: Microscope was equipped with a Cs corrector with two hexapole elements
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.11_2558: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218837 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
14WAT

4wat

1
25TIK

5tik

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0065201
ELECTRON MICROSCOPYf_angle_d1.0866999
ELECTRON MICROSCOPYf_dihedral_angle_d6.6153126
ELECTRON MICROSCOPYf_chiral_restr0.058758
ELECTRON MICROSCOPYf_plane_restr0.006876

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