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- EMDB-7794: Thermostabilized phosphorylated chicken CFTR -

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Basic information

Entry
Database: EMDB / ID: EMD-7794
TitleThermostabilized phosphorylated chicken CFTR
Map dataBinned Sharp map PKA-treated chTS
Sample
  • Cell: CFTRCystic fibrosis transmembrane conductance regulator
    • Protein or peptide: Cystic fibrosis transmembrane conductance regulator
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsCFTR / MEMBRANE PROTEIN
Function / homology
Function and homology information


RHO GTPases regulate CFTR trafficking / RHOQ GTPase cycle / ABC-family proteins mediated transport / Cargo recognition for clathrin-mediated endocytosis / Aggrephagy / Clathrin-mediated endocytosis / Ub-specific processing proteases / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / bicarbonate transport ...RHO GTPases regulate CFTR trafficking / RHOQ GTPase cycle / ABC-family proteins mediated transport / Cargo recognition for clathrin-mediated endocytosis / Aggrephagy / Clathrin-mediated endocytosis / Ub-specific processing proteases / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / bicarbonate transport / chloride transport / chloride channel complex / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / isomerase activity / chloride transmembrane transport / transmembrane transport / recycling endosome membrane / early endosome membrane / apical plasma membrane / endoplasmic reticulum membrane / ATP binding / membrane / plasma membrane / cytosol
Similarity search - Function
CFTR regulator domain / Cystic fibrosis TM conductance regulator (CFTR), regulator domain / Cystic fibrosis transmembrane conductance regulator / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter ...CFTR regulator domain / Cystic fibrosis TM conductance regulator (CFTR), regulator domain / Cystic fibrosis transmembrane conductance regulator / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Cystic fibrosis transmembrane conductance regulator / Cystic fibrosis transmembrane conductance regulator
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsFay JF / Riordan JR
CitationJournal: Biochemistry / Year: 2018
Title: Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.
Authors: Jonathan F Fay / Luba A Aleksandrov / Timothy J Jensen / Liying L Cui / Joseph N Kousouros / Lihua He / Andrei A Aleksandrov / Drew S Gingerich / John R Riordan / James Z Chen /
Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic ...The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.
History
DepositionApr 16, 2018-
Header (metadata) releaseMay 30, 2018-
Map releaseOct 17, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.571
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.571
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6d3s
  • Surface level: 0.571
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7794.png

Downloads & links

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Map

FileDownload / File: emd_7794.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBinned Sharp map PKA-treated chTS
Voxel sizeX=Y=Z: 1.71 Å
Density
Contour LevelBy AUTHOR: 0.571 / Movie #1: 0.571
Minimum - Maximum-0.6483847 - 1.5729154
Average (Standard dev.)0.0082366355 (±0.06934322)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 273.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.711.711.71
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z273.600273.600273.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.6481.5730.008

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Supplemental data

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Half map: half map

Fileemd_7794_half_map_1.map
Annotationhalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: other half map

Fileemd_7794_half_map_2.map
Annotationother half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CFTR

EntireName: CFTRCystic fibrosis transmembrane conductance regulator
Components
  • Cell: CFTRCystic fibrosis transmembrane conductance regulator
    • Protein or peptide: Cystic fibrosis transmembrane conductance regulator
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: CFTR

SupramoleculeName: CFTR / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Cystic fibrosis transmembrane conductance regulator

MacromoleculeName: Cystic fibrosis transmembrane conductance regulator / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ec: 3.6.3.49
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 162.637438 KDa
Recombinant expressionOrganism: Cricetinae (hamsters)
SequenceString: MQRSPLEKAN IFSKLFFRWT KPILKKGYRQ RLELSDIYQI PSADSADNLS EKLEREWDRE LATSKKKPKL INALRRCFFW KFMFYGILL YLGEVTKSVQ PLLLGRIIAS YDPDNSSERS IAYYLGIGLC LLFLVRTLLI HPSIFGLHHI GMQIRIALFS L IYKKTLKL ...String:
MQRSPLEKAN IFSKLFFRWT KPILKKGYRQ RLELSDIYQI PSADSADNLS EKLEREWDRE LATSKKKPKL INALRRCFFW KFMFYGILL YLGEVTKSVQ PLLLGRIIAS YDPDNSSERS IAYYLGIGLC LLFLVRTLLI HPSIFGLHHI GMQIRIALFS L IYKKTLKL SSKVLDKIST GQLVSLLSNN LNKFDEGLAL AHFVWIAPLQ VALLMGLLWD MLQASAFAGL AFLIVMAFFQ AW LGQMMMK YRDKRAGKIN ERLVITSEII ENIQSVKAYC WEDAMEKMIE SLRETELKLT RKAAYVRYFN SSAFFFSGFF VVF LAVVPY AVTKGIILRK IFTTISFCIV LRMTVTRQFP GSVQTWYDSI GAINKIQDFL LKEEYKALEY NLTTTGVEVD KVTA FWDEH ASPVLQDINF KIEKGELLAV SGSTGSGKTS LLMLIMGELE PSEGKIKHSG RISFSPQVSW IMPGTIKENI IFGVS YDEY RYKSVIQACQ LEEDILKFPD KDYTVLGEGG IILSGGQRAR ISLARAVYKD ADLYLMDSPF GYLDIFTEKE IFESCV CKL MANKTRILVT SKLEHLKIAD KILILHEGSC YFYGTFSELQ GQRPDFSSEL MGFDSFDQFS AERRNSIITE TLRRFSF EG ESMGSRNEMK KQSFKQTSDF NDKRKNSIII NPLNAGRKLS IMQKNGTQVN GLEDGHIDSP ERRISLVPDL EQGDVGLP R SNMLNSDHML QSRRRQSVLS LMTGTSVNQG PHVSKKGSTS FRKMSVVPQT NLSSEIDIYT RRLSRDSILD ITDEINEED LKECFTDDAE SMGTVTTWNT YFRYITIHKS LIFVLILCVT IFLLEVAASL VLLLFLQKAA QINATQPENA TSDNPPVIIT DTSSYYMIY IYVGIADTLL AMGIFRGLPL VHTLITVSKT LHQKMVHAVL YAPMSTFNSL KAGGILNRFS KDTAILDDLL P LTVFDLIQ LILIVIGAIT VVSILQPYIF LASVPVIAAF IVLRAYFLHT SQQLKQLESE ARSPIFTHLV TSLKGLWTLR AF GRQPYFE TLFHKALNLH TANWFLYLST LRWFQMRIEM IFVVFFSAVA FISIITTGDG PGRVGIILTL AMNIMGTLQW AVN SSIDVD SLMRSVSRIF KFIDMPTEEM KTIKPQKNNQ FSDALIIENR HVKDEKNWPS GGQMTVTDLT ARYTEGGTAV LENI SFSIS SGQTVGLLGR TGSGKSTLLF AFLRLLNTEG DIQIDGVSWN TVSLQQWRKA FGVIPQKVFI FSGTFRKNLD PYGQW NDEE IWKVAEEVGL KSVIEQFPGQ LDFVLVDGGC VLSHGHKQLM CLARSVLSKA KILLLDEPSA HLDPITSQVI RKTLKH AFA DCTVVLSESR LEAILECQRF LVIEDNKMRQ YESIQKLLSE KSSLRQSGSG GGGGGSLEVL FQGDHHHHHH HHHH

UniProtKB: Cystic fibrosis transmembrane conductance regulator

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
0.15 MNaclSodium chlorideSodium chloride
0.04 MTris-HClTris
0.005 MATP-Mg
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 3410 / Average electron dose: 42.0 e/Å2
Details: I had better girds but the vacuum crashed for the grids in the autoloader.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 700000
Startup modelType of model: NONE
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 57826
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Target criteria: iFSC, elec_dens_fast
Output model

PDB-6d3s:
Thermostabilized phosphorylated chicken CFTR

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