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- PDB-6ukj: Single-Particle Cryo-EM Structure of Plasmodium falciparum Chloro... -

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Basic information

Entry
Database: PDB / ID: 6ukj
TitleSingle-Particle Cryo-EM Structure of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform
Components
  • Chloroquine resistance transporter
  • Fab Heavy ChainFragment antigen-binding
  • Fab Light ChainFragment antigen-binding
KeywordsMEMBRANE PROTEIN / Transporter / drug resistance / digestive vacuole of malaria parasite / nanodisc
Function / homologyChloroquine-resistance transporter-like / Chloroquine resistance transporter / CRT-like, chloroquine-resistance transporter-like / xenobiotic transmembrane transporter activity / integral component of membrane / Chloroquine resistance transporter
Function and homology information
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKim, J. / Tan, Y.Z. / Wicht, K.J. / Erramilli, S.K. / Dhingra, S.K. / Okombo, J. / Vendome, J. / Hagenah, L.M. / Giacometti, S.I. / Warren, A.L. / Nosol, K. / Roepe, P.D. / Potter, C.S. / Carragher, B. / Kossiakoff, A.A. / Quick, M. / Fidock, D.A. / Mancia, F.
Funding support United States, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01 GM111980 United States
National Institutes of Health/National Institute of General Medical SciencesR37 AI50234 United States
National Institutes of Health/National Institute of General Medical SciencesR01 AI124678 United States
National Institutes of Health/National Institute of General Medical SciencesR01 GM119396 United States
National Institutes of Health/National Institute of General Medical SciencesR01 AI506312 United States
National Institutes of Health/National Institute of General Medical SciencesAI111962 United States
National Institutes of Health/National Institute of General Medical SciencesT32 HL120826 United States
National Institutes of Health/National Institute of General Medical SciencesP41 GM103310 United States
National Institutes of Health/National Institute of General Medical SciencesP41 GM116799 United States
CitationJournal: Nature / Year: 2019
Title: Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.
Authors: Jonathan Kim / Yong Zi Tan / Kathryn J Wicht / Satchal K Erramilli / Satish K Dhingra / John Okombo / Jeremie Vendome / Laura M Hagenah / Sabrina I Giacometti / Audrey L Warren / Kamil Nosol / Paul D Roepe / Clinton S Potter / Bridget Carragher / Anthony A Kossiakoff / Matthias Quick / David A Fidock / Filippo Mancia /
Abstract: The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and ...The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Chloroquine resistance transporter
H: Fab Heavy Chain
L: Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,6554
Polymers102,1683
Non-polymers4871
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, PfCRT and Fab complex shifts the gel filtration peak leftwards compared to PfCRT alone.
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5370 Å2
ΔGint-26 kcal/mol
Surface area26280 Å2

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Components

#1: Protein/peptide Chloroquine resistance transporter


Mass: 53074.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate 7G8) (eukaryote)
Strain: isolate 7G8 / Gene: PFBG_01689 / Cell line (production host): HEK 293 F / Production host: Homo sapiens (human) / References: UniProt: W7FI62
#2: Protein/peptide Fab Heavy Chain / Fragment antigen-binding


Mass: 25737.631 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Protein/peptide Fab Light Chain / Fragment antigen-binding


Mass: 23355.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestY

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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

Component

Source: RECOMBINANT / Type: ORGANELLE OR CELLULAR COMPONENT

IDNameEntity IDParent-ID
1Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab1, 2, 30
2Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)11
3Antigen-binding fragment (Fab)2, 31
Molecular weight

Entity assembly-ID: 1 / Experimental value: NO

IDValue (°)
1
20.049 MDa
30.050 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Plasmodium falciparum 7G8 (eukaryote)57266
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606HEK 293 F
33Escherichia coli (E. coli)562
Buffer solutionpH: 7 / Details: Solution was filtered and degassed.
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPESC8H18N2O4S
2150 mMSodium ChlorideNaClSodium chloride
SpecimenConc.: 1.56 mg/ml
Details: Protein was incorporated into lipid nanodiscs and complexed with Fab.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 279 K / Details: Blot for 2 seconds before plunging.

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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: 215000 X / Nominal defocus max: 1.8 nm / Nominal defocus min: 1.2 nm / Cs: 0.001 mm / Alignment procedure: OTHER
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 91.56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3377
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Spherical aberration corrector: Cs corrector was used.
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 80 / Used frames/image: 1-80

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Processing

EM software
IDNameVersionCategory
2Gautomatch0.53particle selection
3Leginon3.4image acquisition
5RELION2.1CTF correction
6cisTEM1CTF correction
7cryoSPARC2CTF correction
10Cootmodel fitting
12PHENIXmodel refinement
13cryoSPARC2initial Euler assignment
14cryoSPARC2final Euler assignment
15cryoSPARC2classification
16cisTEM13D reconstruction
Image processingDetails: Energy filter slit width of 20 eV was used during the collection and was aligned automatically every hour using Leginon.
CTF correctionDetails: CTF was estimated using GCTF and refined throughout the pipeline using cisTEM.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 183241
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17030 / Algorithm: BACK PROJECTION
Details: CisTEM was used to reconstruct the final map using original (non-signal subtracted) particles.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Rosetta was used to generate an ab initio model for the PfCRT using the map. 4XMM chains H and L were used as starting point to model the Fab.
Atomic model building

3D fitting-ID: 1 / PDB-ID: 4XMM

IDPdb chain-ID
1H
2L

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