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- EMDB-45193: V-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild... -

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Basic information

Entry
Database: EMDB / ID: EMD-45193
TitleV-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (sharpened map from cryoSPARC non-uniform refinement)
Map dataV-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (sharpened map from cryoSPARC non-uniform refinement)
Sample
  • Complex: Wild-type human CFTR solubilized in digitonin and cholesterol-hemisuccinate plus T2a nanobody in the presence of 2 mM MgATP
    • Organelle or cellular component: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)
      • Organelle or cellular component: T2a nanobody
        • Protein or peptide: T2a nanobody
      • Protein or peptide: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)
Keywordscystic fibrosis / CFTR / nanobody / ATPase / conformational ensemble / MEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of voltage-gated chloride channel activity / : / Sec61 translocon complex binding / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / positive regulation of enamel mineralization / transepithelial water transport / RHO GTPases regulate CFTR trafficking / amelogenesis / intracellular pH elevation ...positive regulation of voltage-gated chloride channel activity / : / Sec61 translocon complex binding / channel-conductance-controlling ATPase / intracellularly ATP-gated chloride channel activity / positive regulation of enamel mineralization / transepithelial water transport / RHO GTPases regulate CFTR trafficking / amelogenesis / intracellular pH elevation / chloride channel inhibitor activity / : / Golgi-associated vesicle membrane / multicellular organismal-level water homeostasis / cholesterol transport / bicarbonate transport / bicarbonate transmembrane transporter activity / chloride channel regulator activity / vesicle docking involved in exocytosis / membrane hyperpolarization / chloride transmembrane transporter activity / sperm capacitation / cholesterol biosynthetic process / RHOQ GTPase cycle / chloride channel activity / positive regulation of exocytosis / ATPase-coupled transmembrane transporter activity / chloride channel complex / ABC-type transporter activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / 14-3-3 protein binding / cellular response to forskolin / chloride transmembrane transport / response to endoplasmic reticulum stress / cellular response to cAMP / PDZ domain binding / establishment of localization in cell / clathrin-coated endocytic vesicle membrane / Defective CFTR causes cystic fibrosis / Late endosomal microautophagy / recycling endosome / ABC-family proteins mediated transport / transmembrane transport / recycling endosome membrane / Chaperone Mediated Autophagy / Aggrephagy / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / protein-folding chaperone binding / early endosome membrane / early endosome / endosome membrane / Ub-specific processing proteases / apical plasma membrane / lysosomal membrane / endoplasmic reticulum membrane / enzyme binding / cell surface / protein-containing complex / ATP hydrolysis activity / ATP binding / nucleus / membrane / plasma membrane / cytoplasm / 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 ...: / 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
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsHunt JF / Paige AS / Cohen BM / Goldberg PM / Wang C / Loughlin BJ / Kappes JC / Yang Z / Jiang F / Govaerts C ...Hunt JF / Paige AS / Cohen BM / Goldberg PM / Wang C / Loughlin BJ / Kappes JC / Yang Z / Jiang F / Govaerts C / Overtus M / Rich Z
Funding support United States, 1 items
OrganizationGrant numberCountry
Cystic Fibrosis FoundationHUNT13XX0,HUNT18G0,HUNT20G0,004400G222,FRANK16XX0,FRANK18G0 United States
CitationJournal: To Be Published
Title: V-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (sharpened map from cryoSPARC non-uniform refinement)
Authors: Hunt FJ / Paige AS / Cohen BM / Goldberg PM / Wang C / Loughlin BJ / Kappes JC / Yang Z / Jiang F / Govaerts C / Overtus M / Urbatsch IL / Lukacs G
History
DepositionJun 5, 2024-
Header (metadata) releaseJul 2, 2025-
Map releaseJul 2, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45193.png

Downloads & links

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Map

FileDownload / File: emd_45193.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationV-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (sharpened map from cryoSPARC non-uniform refinement)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 267.2 Å		0.84 Å/pix.
x 320 pix.
= 267.2 Å		0.84 Å/pix.
x 320 pix.
= 267.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.144
Minimum - Maximum-0.5491271 - 0.9856996
Average (Standard dev.)0.0005771722 (±0.022749)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: V-Shaped, Channel Formed, T2a Nanobody Bound conformation of...

Fileemd_45193_half_map_1.map
AnnotationV-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (half map B from cryoSPARC non-uniform refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: V-Shaped, Channel Formed, T2a Nanobody Bound conformation of...

Fileemd_45193_half_map_2.map
AnnotationV-Shaped, Channel Formed, T2a Nanobody Bound conformation of wild-type human CFTR (half map B from cryoSPARC non-uniform refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Wild-type human CFTR solubilized in digitonin and cholesterol-hem...

EntireName: Wild-type human CFTR solubilized in digitonin and cholesterol-hemisuccinate plus T2a nanobody in the presence of 2 mM MgATP
Components
  • Complex: Wild-type human CFTR solubilized in digitonin and cholesterol-hemisuccinate plus T2a nanobody in the presence of 2 mM MgATP
    • Organelle or cellular component: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)
      • Organelle or cellular component: T2a nanobody
        • Protein or peptide: T2a nanobody
      • Protein or peptide: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)

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Supramolecule #1: Wild-type human CFTR solubilized in digitonin and cholesterol-hem...

SupramoleculeName: Wild-type human CFTR solubilized in digitonin and cholesterol-hemisuccinate plus T2a nanobody in the presence of 2 mM MgATP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 15.8 KDa

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Supramolecule #2: Wild type human Cystic Fibrosis Transmembrane Conductance Regulat...

SupramoleculeName: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)
type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: T2a nanobody

SupramoleculeName: T2a nanobody / type: organelle_or_cellular_component / ID: 3 / Parent: 2 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Wild type human Cystic Fibrosis Transmembrane Conductance Regulat...

MacromoleculeName: Wild type human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR)
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MQRSPLEKAS VVSKLFFSWT RPILRKGYRQ RLELSDIYQI PSVDSADNLS EKLEREWDRE LASKKNPKLI NALRRCFFWR FMFYGIFLYL GEVTKAVQPL LLGRIIASYD PDNKEERSIA IYLGIGLCLL FIVRTLLLHP AIFGLHHIGM QMRIAMFSLI YKKTLKLSSR ...String:
MQRSPLEKAS VVSKLFFSWT RPILRKGYRQ RLELSDIYQI PSVDSADNLS EKLEREWDRE LASKKNPKLI NALRRCFFWR FMFYGIFLYL GEVTKAVQPL LLGRIIASYD PDNKEERSIA IYLGIGLCLL FIVRTLLLHP AIFGLHHIGM QMRIAMFSLI YKKTLKLSSR VLDKISIGQL VSLLSNNLNK FDEGLALAHF VWIAPLQVAL LMGLIWELLQ ASAFCGLGFL IVLALFQAGL GRMMMKYRDQ RAGKISERLV ITSEMIENIQ SVKAYCWEEA MEKMIENLRQ TELKLTRKAA YVRYFNSSAF FFSGFFVVFL SVLPYALIKG IILRKIFTTI SFCIVLRMAV TRQFPWAVQT WYDSLGAINK IQDFLQKQEY KTLEYNLTTT EVVMENVTAF WEEGFGELFE KAKQNNNNRK TSNGDDSLFF SNFSLLGTPV LKDINFKIER GQLLAVAGST GAGKTSLLMM IMGELEPSEG KIKHSGRISF CSQFSWIMPG TIKENIIFGV SYDEYRYRSV IKACQLEEDI SKFAEKDNIV LGEGGITLSG GQRARISLAR AVYKDADLYL LDSPFGYLDV LTEKEIFESC VCKLMANKTR ILVTSKMEHL KKADKILILH EGSSYFYGTF SELQNLQPDF SSKLMGCDSF DQFSAERRNS ILTETLHRFS LEGDAPVSWT ETKKQSFKQT GEFGEKRKNS ILNPINSIRK FSIVQKTPLQ MNGIEEDSDE PLERRLSLVP DSEQGEAILP RISVISTGPT LQARRRQSVL NLMTHSVNQG QNIHRKTTAS TRKVSLAPQA NLTELDIYSR RLSQETGLEI SEEINEEDLK ECFFDDMESI PAVTTWNTYL RYITVHKSLI FVLIWCLVIF LAEVAASLVV LWLLGNTPLQ DKGNSTHSRN NSYAVIITST SSYYVFYIYV GVADTLLAMG FFRGLPLVHT LITVSKILHH KMLHSVLQAP MSTLNTLKAG GILNRFSKDI AILDDLLPLT IFDFIQLLLI VIGAIAVVAV LQPYIFVATV PVIVAFIMLR AYFLQTSQQL KQLESEGRSP IFTHLVTSLK GLWTLRAFGR QPYFETLFHK ALNLHTANWF LYLSTLRWFQ MRIEMIFVIF FIAVTFISIL TTGEGEGRVG IILTLAMNIM STLQWAVNSS IDVDSLMRSV SRVFKFIDMP TEGKPTKSTK PYKNGQLSKV MIIENSHVKK DDIWPSGGQM TVKDLTAKYT EGGNAILENI SFSISPGQRV GLLGRTGSGK STLLSAFLRL LNTEGEIQID GVSWDSITLQ QWRKAFGVIP QKVFIFSGTF RKNLDPYEQW SDQEIWKVAD EVGLRSVIEQ FPGKLDFVLV DGGCVLSHGH KQLMCLARSV LSKAKILLLD EPSAHLDPVT YQIIRRTLKQ AFADCTVILC EHRIEAMLEC QQFLVIEENK VRQYDSIQKL LNERSLFRQA ISPSDRVKLF PHRNSSKCKS KPQIAALKEE TEEEVQDTRL LEENLYFQGG GGSGGSWSHP QFEKAAAGGG SGGGSWSHPQ FEK

UniProtKB: Cystic fibrosis transmembrane conductance regulator

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Macromolecule #2: T2a nanobody

MacromoleculeName: T2a nanobody / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGSIFR IDAMGWYRQA PGKQRELVAH STSGGSTDYA DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT AVYYCNADVR TRWYASNN Y WGQGTQVTVS SAAAHHHHHH GAAEQKLISE EDLNGAA

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
3.0 mMMgCl2Magnesium Chloride
50.0 mMC4H11NO3Tris buffer
0.06 % (w/v)C56H92O29Digitonin
2.0 mMC10H16N5O13P3ATP
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.026000000000000002 kPa
Details: Gatan Solaris Plasma Cleaner 950, 15 watts cleaning power, O2/H2 at 27.5/6.4 sccm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 85.0 K / Max: 90.0 K
Specialist opticsEnergy filter - Name: GIF Quantum ER / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 11110 / Average exposure time: 2.5 sec. / Average electron dose: 58.06 e/Å2
Details: Movies comprised 50 frames collected in 2.5 seconds.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2635244
CTF correctionSoftware - Name: cryoSPARC (ver. 4.3.1) / Software - details: Patch CTF correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 102624
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final 3D classificationNumber classes: 16 / Avg.num./class: 29000 / Software - Name: cryoSPARC (ver. 4.3.1)
Details: Final cycle of iterative heterorefinement against 11 protein volumes and 4 dummy volumes.
FSC plot (resolution estimation)

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