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- EMDB-20806: Single-Particle Cryo-EM Structure of Plasmodium falciparum Chloro... -

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Basic information

Entry
Database: EMDB / ID: EMD-20806
TitleSingle-Particle Cryo-EM Structure of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform
Map data
SamplePlasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
  • Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)
  • Antigen-binding fragment (Fab)
  • Chloroquine resistance transporter
  • Fab Heavy ChainFragment antigen-binding
  • Fab Light ChainFragment antigen-binding
  • ligand
Function / homologyChloroquine-resistance transporter-like / Chloroquine resistance transporter / xenobiotic transmembrane transporter activity / integral component of membrane / Chloroquine resistance transporter
Function and homology information
Biological speciesPlasmodium falciparum 7G8 (eukaryote) / Homo sapiens (human) / Plasmodium falciparum (isolate 7G8) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKim J / Tan YZ / Wicht KJ / Erramilli SK / Dhingra SK / Okombo J / Vendome J / Hagenah LM / Giacometti SI / Warren AL / Nosol K / Roepe PD / Potter CS / Carragher B / Kossiakoff AA / Quick M / Fidock DA / Mancia F
Funding support United States, 9 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01 GM111980 United States
National Institutes of Health/National Institute of General Medical SciencesR01 AI124678 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 AI506312 United States
National Institutes of Health/National Institute of General Medical SciencesR01 GM119396 United States
National Institutes of Health/National Institute of General Medical SciencesT32 HL120826 United States
National Institutes of Health/National Institute of General Medical SciencesAI111962 United States
National Institutes of Health/National Institute of General Medical SciencesP41 GM116799 United States
National Institutes of Health/National Institute of General Medical SciencesP41 GM103310 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 ReportPDB-ID: 6ukj

SummaryFull reportAbout validation report
History
DepositionOct 5, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseDec 4, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6ukj
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20806.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.04 Å/pix.
x 300 pix.
= 310.5 Å
1.04 Å/pix.
x 300 pix.
= 310.5 Å
1.04 Å/pix.
x 300 pix.
= 310.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.035 Å
Density
Contour LevelBy AUTHOR: 8 / Movie #1: 8
Minimum - Maximum-18.20441 - 42.020454
Average (Standard dev.)-0.00000000000 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 310.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0351.0351.035
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z310.500310.500310.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-18.20442.020-0.000

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Supplemental data

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Segmentation: #1

Fileemd_20806_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Segmentation: #2

Fileemd_20806_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) ...

EntireName: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
Number of components: 7

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Component #1: cellular-component, Plasmodium falciparum Chloroquine Resistance ...

Cellular-componentName: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
Recombinant expression: No
MassTheoretical: 50 kDa

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Component #2: cellular-component, Plasmodium falciparum Chloroquine Resistance ...

Cellular-componentName: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)
Recombinant expression: No
SourceSpecies: Plasmodium falciparum 7G8 (eukaryote)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK 293 F

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Component #3: cellular-component, Antigen-binding fragment (Fab)

Cellular-componentName: Antigen-binding fragment (Fab) / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Chloroquine resistance transporter

ProteinName: Chloroquine resistance transporter / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 53.074461 kDa
SourceSpecies: Plasmodium falciparum (isolate 7G8) (eukaryote) / Strain: isolate 7G8
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Fab Heavy Chain

ProteinName: Fab Heavy ChainFragment antigen-binding / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.737631 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Fab Light Chain

ProteinName: Fab Light ChainFragment antigen-binding / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.355898 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: ligand, CHOLESTEROL HEMISUCCINATE

LigandName: CHOLESTEROL HEMISUCCINATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.486726 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.56 mg/mL / Buffer solution: Solution was filtered and degassed. / pH: 7
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 279 K / Humidity: 80 % / Details: Blot for 2 seconds before plunging..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 91.56 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 215000.0 X (nominal) / Cs: 0.001 mm / Imaging mode: BRIGHT FIELD / Defocus: 1.2 - 1.8 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3377 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 17030
Details: Energy filter slit width of 20 eV was used during the collection and was aligned automatically every hour using Leginon.
3D reconstructionAlgorithm: BACK PROJECTION / Software: cisTEM
CTF correction: CTF was estimated using GCTF and refined throughout the pipeline using cisTEM.
Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF
Details: CisTEM was used to reconstruct the final map using original (non-signal subtracted) particles.
Euler angles: Non-uniform refinement in CryoSPARC was used.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement 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.
Input PDB model: 4XMM, 4XMM
Chain ID: H, L
Output model

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