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

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Entry
Database: EMDB / ID: EMD-20806
TitleSingle-Particle Cryo-EM Structure of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform
Map dataSharpened map
Sample
  • Organelle or cellular component: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
    • Organelle or cellular component: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)
      • Protein or peptide: Chloroquine resistance transporter
    • Organelle or cellular component: Antigen-binding fragment (Fab)
      • Protein or peptide: Fab Heavy ChainFragment antigen-binding
      • Protein or peptide: Fab Light ChainFragment antigen-binding
  • Ligand: CHOLESTEROL HEMISUCCINATE
Function / homologyChloroquine-resistance transporter-like / Chloroquine resistance transporter / CRT-like, chloroquine-resistance transporter-like / vacuolar membrane / xenobiotic transmembrane transporter activity / membrane => GO:0016020 / 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 ...Kim 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 Sciences (NIH/NIGMS)R01 GM111980 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 AI124678 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R37 AI50234 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 AI506312 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM119396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 HL120826 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)AI111962 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM116799 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 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 ...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.
History
DepositionOct 5, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseDec 4, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing 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
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  • 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20806.png

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)
AnnotationSharpened map
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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Mask #1

Fileemd_20806_msk_1.map
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AxesZYX

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Mask #2

Fileemd_20806_msk_2.map
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Additional map: Unsharpened map

Fileemd_20806_additional_1.map
AnnotationUnsharpened map
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Additional map: Local resolution map

Fileemd_20806_additional_2.map
AnnotationLocal resolution map
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Additional map: 3DFSC - PfCRT with Fab, Raw

Fileemd_20806_additional_3.map
Annotation3DFSC - PfCRT with Fab, Raw
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Additional map: 3DFSC - PfCRT with Fab, Thresholded

Fileemd_20806_additional_4.map
Annotation3DFSC - PfCRT with Fab, Thresholded
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Additional map: 3DFSC - PfCRT with Fab, Thresholded and Binarized

Fileemd_20806_additional_5.map
Annotation3DFSC - PfCRT with Fab, Thresholded and Binarized
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Additional map: 3DFSC - PfCRT only, Raw

Fileemd_20806_additional_6.map
Annotation3DFSC - PfCRT only, Raw
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Additional map: 3DFSC - PfCRT Only, Thresholded

Fileemd_20806_additional_7.map
Annotation3DFSC - PfCRT Only, Thresholded
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Additional map: 3DFSC - PfCRT only, Thresholded and binarized

Fileemd_20806_additional_8.map
Annotation3DFSC - PfCRT only, Thresholded and binarized
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AxesZYX

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Half map: Half map 1

Fileemd_20806_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_20806_half_map_2.map
AnnotationHalf map 2
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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
Components
  • Organelle or cellular component: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
    • Organelle or cellular component: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)
      • Protein or peptide: Chloroquine resistance transporter
    • Organelle or cellular component: Antigen-binding fragment (Fab)
      • Protein or peptide: Fab Heavy ChainFragment antigen-binding
      • Protein or peptide: Fab Light ChainFragment antigen-binding
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) ...

SupramoleculeName: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform Complexed with Fab
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 102 KDa

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Supramolecule #2: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

SupramoleculeName: Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)
type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Plasmodium falciparum 7G8 (eukaryote)
Molecular weightTheoretical: 49 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293 F

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Supramolecule #3: Antigen-binding fragment (Fab)

SupramoleculeName: Antigen-binding fragment (Fab) / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Chloroquine resistance transporter

MacromoleculeName: Chloroquine resistance transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Plasmodium falciparum (isolate 7G8) (eukaryote) / Strain: isolate 7G8
Molecular weightTheoretical: 53.074461 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGRAKFASKK NNQKNSSKND ERYRELDNLV QEGNGSRLGG GSCLGKCAHV FKLIFKEIKD NIFIYILSII YLSVSVMNTI FAKRTLNKI GNYSFVTSET HNFICMIMFF IVYSLFGNKK GNSKERHRSF NLQFFAISML DACSVILAFI GLTRTTGNIQ S FVLQLSIP ...String:
MGRAKFASKK NNQKNSSKND ERYRELDNLV QEGNGSRLGG GSCLGKCAHV FKLIFKEIKD NIFIYILSII YLSVSVMNTI FAKRTLNKI GNYSFVTSET HNFICMIMFF IVYSLFGNKK GNSKERHRSF NLQFFAISML DACSVILAFI GLTRTTGNIQ S FVLQLSIP INMFFCFLIL RYRYHLYNYL GAVIIVVTIA LVEMKLSFET QEENSIIFNL VLISSLIPVC FSNMTREIVF KK YKIDILR LNAMVSFFQL FTSCLILPVY TLPFLKQLHL PYNEIWTNIK NGFACLFLGR NTVVENCGLG MAKLCDDCDG AWK TFALFS FFDICDNLIT SYIIDKFSTM TYTIVSCIQG PALAIAYYFK FLAGDVVREP RLLDFVTLFG YLFGSIIYRV GNII LERKK MRNEENEDSE GELTNVDSII TQAAAGGGSG GGSENLYFQG SHHHHHHHHH HWSHPQFEK

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Macromolecule #2: Fab Heavy Chain

MacromoleculeName: Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.737631 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSYSYIHWV RQAPGKGLEW VASIYPYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYGSNYSFW YRGSSVTYAI DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSYSYIHWV RQAPGKGLEW VASIYPYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYGSNYSFW YRGSSVTYAI DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HT C

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Macromolecule #3: Fab Light Chain

MacromoleculeName: Fab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.355898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSTWPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSTWPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.56 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium Chloride

Details: Solution was filtered and degassed.
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 279 K / Instrument: LEICA EM GP / Details: Blot for 2 seconds before plunging..
DetailsProtein was incorporated into lipid nanodiscs and complexed with Fab.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 0.0018 µm / Nominal defocus min: 0.0012 µm / Nominal magnification: 215000
Specialist opticsSpherical aberration corrector: Cs corrector was used. / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-80 / Number grids imaged: 1 / Number real images: 3377 / Average exposure time: 6.0 sec. / Average electron dose: 91.56 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 183241
CTF correctionSoftware: (Name: RELION (ver. 2.1), cisTEM (ver. 1), cryoSPARC (ver. 2))
Details: CTF was estimated using GCTF and refined throughout the pipeline using cisTEM.
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final 3D classificationNumber classes: 2 / Avg.num./class: 17841 / Software - Name: cryoSPARC (ver. 2)
Details: Ab initio refinement using 2 models in CryoSPARC was used for classification.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1)
Details: CisTEM was used to reconstruct the final map using original (non-signal subtracted) particles.
Number images used: 17030
DetailsEnergy filter slit width of 20 eV was used during the collection and was aligned automatically every hour using Leginon.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4xmm

chain_id: H

4xmm

chain_id: L
DetailsRosetta 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6ukj:
Single-Particle Cryo-EM Structure of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) 7G8 Isoform

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