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- EMDB-45604: cryo-EM structure of calcineurin-fused beta2 adrenergic receptor ... -

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Basic information

Entry
Database: EMDB / ID: EMD-45604
Titlecryo-EM structure of calcineurin-fused beta2 adrenergic receptor in carazolol bound inactive state
Map data
Sample
  • Complex: calcineurin fused beta2AR in complex with FKBP12
    • Complex: calcineurin fused beta2AR
      • Protein or peptide: Beta-2 adrenergic receptor,Calcineurin subunit B type 1
    • Complex: Protein phosphatase 3 catalytic subunit alpha
      • Protein or peptide: Protein phosphatase 3 catalytic subunit alpha
    • Complex: FKBP1A
      • Protein or peptide: Peptidyl-prolyl cis-trans isomerase FKBP1A
  • Ligand: (2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-ol
  • Ligand: 8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN
KeywordsGPCR / cryo-EM / calcineurin fusion / inactive state / MEMBRANE PROTEIN / MEMBRANE PROTEIN-HYDROLASE-ISOMERASE complex
Function / homology
Function and homology information


negative regulation of angiotensin-activated signaling pathway / calcium-dependent protein serine/threonine phosphatase regulator activity / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / CLEC7A (Dectin-1) induces NFAT activation / negative regulation of signaling / calcium-dependent protein serine/threonine phosphatase activity / Calcineurin activates NFAT / positive regulation of cardiac muscle hypertrophy in response to stress ...negative regulation of angiotensin-activated signaling pathway / calcium-dependent protein serine/threonine phosphatase regulator activity / regulation of cell proliferation involved in kidney morphogenesis / positive regulation of glomerulus development / negative regulation of calcium ion import across plasma membrane / CLEC7A (Dectin-1) induces NFAT activation / negative regulation of signaling / calcium-dependent protein serine/threonine phosphatase activity / Calcineurin activates NFAT / positive regulation of cardiac muscle hypertrophy in response to stress / positive regulation of saliva secretion / positive regulation of calcium ion import across plasma membrane / negative regulation of dendrite morphogenesis / calcineurin complex / positive regulation of connective tissue replacement / slit diaphragm / protein serine/threonine phosphatase complex / FCERI mediated Ca+2 mobilization / Ca2+ pathway / macrolide binding / activin receptor binding / renal filtration / lung epithelial cell differentiation / calcineurin-NFAT signaling cascade / regulation of skeletal muscle contraction by regulation of release of sequestered calcium ion / cytoplasmic side of membrane / transforming growth factor beta receptor binding / TGFBR1 LBD Mutants in Cancer / positive regulation of calcineurin-NFAT signaling cascade / transition between fast and slow fiber / myelination in peripheral nervous system / type I transforming growth factor beta receptor binding / negative regulation of activin receptor signaling pathway / heart trabecula formation / positive regulation of osteoclast differentiation / I-SMAD binding / positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / cardiac muscle hypertrophy in response to stress / AMPA selective glutamate receptor signaling pathway / signaling receptor inhibitor activity / regulation of amyloid precursor protein catabolic process / regulation of synaptic vesicle cycle / norepinephrine binding / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / positive regulation of autophagosome maturation / heat generation / terminal cisterna / Adrenoceptors / ryanodine receptor complex / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction / positive regulation of lipophagy / : / dendrite morphogenesis / 'de novo' protein folding / negative regulation of multicellular organism growth / negative regulation of G protein-coupled receptor signaling pathway / ventricular cardiac muscle tissue morphogenesis / CLEC7A (Dectin-1) induces NFAT activation / adrenergic receptor signaling pathway / histone H2AXS140 phosphatase activity / RNA polymerase II CTD heptapeptide repeat Y1 phosphatase activity / RNA polymerase II CTD heptapeptide repeat T4 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S2 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S5 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S7 phosphatase activity / MAP kinase serine/threonine phosphatase activity / response to psychosocial stress / calmodulin-dependent protein phosphatase activity / myosin phosphatase activity / protein-serine/threonine phosphatase / branching involved in blood vessel morphogenesis / endosome to lysosome transport / FK506 binding / positive regulation of cardiac muscle hypertrophy / regulation of postsynaptic neurotransmitter receptor internalization / parallel fiber to Purkinje cell synapse / channel regulator activity / protein peptidyl-prolyl isomerization / diet induced thermogenesis / neuronal dense core vesicle / positive regulation of cAMP/PKA signal transduction / positive regulation of activated T cell proliferation / positive regulation of endocytosis / TGF-beta receptor signaling activates SMADs / mTORC1-mediated signalling / phosphoprotein phosphatase activity / adenylate cyclase binding / Calcineurin activates NFAT / regulation of ryanodine-sensitive calcium-release channel activity / DARPP-32 events / smooth muscle contraction / Activation of BAD and translocation to mitochondria / epidermis development / epithelial to mesenchymal transition / bone resorption / regulation of immune response / phosphatase binding / postsynaptic modulation of chemical synaptic transmission
Similarity search - Function
PP2B, metallophosphatase domain / PP2B / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Beta 2 adrenoceptor / Adrenoceptor family / : / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase ...PP2B, metallophosphatase domain / PP2B / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Beta 2 adrenoceptor / Adrenoceptor family / : / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase / Peptidyl-prolyl cis-trans isomerase domain superfamily / Serpentine type 7TM GPCR chemoreceptor Srsx / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / G-protein coupled receptors family 1 signature. / EF-hand domain pair / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Beta-2 adrenergic receptor / Peptidyl-prolyl cis-trans isomerase FKBP1A / Calcineurin subunit B type 1 / Protein phosphatase 3 catalytic subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsXu J / Chen G / Du Y / Kobilka BK
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR.
Authors: Jun Xu / Geng Chen / Haoqing Wang / Sheng Cao / Jie Heng / Xavier Deupi / Yang Du / Brian K Kobilka /
Abstract: Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G ...Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here, we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6, and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β adrenergic receptor (βAR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.
History
DepositionJul 2, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45604.png

Downloads & links

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Map

FileDownload / File: emd_45604.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-3.567482 - 4.6001105
Average (Standard dev.)0.009945005 (±0.07515255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_45604_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_45604_half_map_2.map
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Sample components

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Entire : calcineurin fused beta2AR in complex with FKBP12

EntireName: calcineurin fused beta2AR in complex with FKBP12
Components
  • Complex: calcineurin fused beta2AR in complex with FKBP12
    • Complex: calcineurin fused beta2AR
      • Protein or peptide: Beta-2 adrenergic receptor,Calcineurin subunit B type 1
    • Complex: Protein phosphatase 3 catalytic subunit alpha
      • Protein or peptide: Protein phosphatase 3 catalytic subunit alpha
    • Complex: FKBP1A
      • Protein or peptide: Peptidyl-prolyl cis-trans isomerase FKBP1A
  • Ligand: (2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-ol
  • Ligand: 8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN

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Supramolecule #1: calcineurin fused beta2AR in complex with FKBP12

SupramoleculeName: calcineurin fused beta2AR in complex with FKBP12 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: calcineurin fused beta2AR

SupramoleculeName: calcineurin fused beta2AR / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Protein phosphatase 3 catalytic subunit alpha

SupramoleculeName: Protein phosphatase 3 catalytic subunit alpha / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #4: FKBP1A

SupramoleculeName: FKBP1A / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Beta-2 adrenergic receptor,Calcineurin subunit B type 1

MacromoleculeName: Beta-2 adrenergic receptor,Calcineurin subunit B type 1
type: protein_or_peptide / ID: 1
Details: residues 29-354 (Uniprot numbering) of the beta2-AR with the third cytoplasmic domain replaced with residues 16-170 (Uniprot numbering) of calcineurin subunit B
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.309711 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DEVWVVGMGI VMSLIVLAIV FGNVLVITAI AKFERLQTVT NYFITSLACA DLVMGLAVVP FGAAHILMKM WTFGNFWCEF WTSIDVLCV TASIETLCVI AVDRYFAITS PFKYQSLLTK NKARVIILMV WIVSGLTSFL PIQMHWYRAT HQEAINCYAE E TCCDFFTN ...String:
DEVWVVGMGI VMSLIVLAIV FGNVLVITAI AKFERLQTVT NYFITSLACA DLVMGLAVVP FGAAHILMKM WTFGNFWCEF WTSIDVLCV TASIETLCVI AVDRYFAITS PFKYQSLLTK NKARVIILMV WIVSGLTSFL PIQMHWYRAT HQEAINCYAE E TCCDFFTN QAYAIASSIV SFYVPLVIMV FVYSRVFQEA KRQLDADEIK RLGKRFKKLD LDNSGSLSVE EFMSLPELQQ NP LVQRVID IFDTDGNGEV DFKEFIEGVS QFSVKGDKEQ KLRFAFRIYD MDKDGYISNG ELFQVLKMMV GNNLKDTQLQ QIV DKTIIN ADKDGDGRIS FEEFCAVVGG LDIHKKMVVD VKFCLKEHKA LKTLGIIMGT FTLCWLPFFI VNIVHVIQDN LIRK EVYIL LNWIGYVNSG FNPLIYCRSP DFRIAFQELL CLRRDDLKAY GNGY

UniProtKB: Beta-2 adrenergic receptor, Calcineurin subunit B type 1, Beta-2 adrenergic receptor

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Macromolecule #2: Protein phosphatase 3 catalytic subunit alpha

MacromoleculeName: Protein phosphatase 3 catalytic subunit alpha / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 42.627785 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SEPKAIDPKL STTDRVVKAV PFPPSHRLTA KEVFDNDGKP RVDILKAHLM KEGRLEESVA LRIITEGASI LRQEKNLLDI DAPVTVCGD IHGQFFDLMK LFEVGGSPAN TRYLFLGDYV DRGYFSIECV LYLWALKILY PKTLFLLRGN HECRHLTEYF T FKQECKIK ...String:
SEPKAIDPKL STTDRVVKAV PFPPSHRLTA KEVFDNDGKP RVDILKAHLM KEGRLEESVA LRIITEGASI LRQEKNLLDI DAPVTVCGD IHGQFFDLMK LFEVGGSPAN TRYLFLGDYV DRGYFSIECV LYLWALKILY PKTLFLLRGN HECRHLTEYF T FKQECKIK YSERVYDACM DAFDCLPLAA LMNQQFLCVH GGLSPEINTL DDIRKLDRFK EPPAYGPMCD ILWSDPLEDF GN EKTQEHF THNTVRGCSY FYSYPAVCDF LQHNNLLSIL RAHEAQDAGY RMYRKSQTTG FPSLITIFSA PNYLDVYNNK AAV LKYENN VMNIRQFNCS PHPYWLPNFM DVFTWSLPFV GEKVTEMLVN VLNI

UniProtKB: Protein phosphatase 3 catalytic subunit alpha

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Macromolecule #3: Peptidyl-prolyl cis-trans isomerase FKBP1A

MacromoleculeName: Peptidyl-prolyl cis-trans isomerase FKBP1A / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidylprolyl isomerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.967705 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFM LGKQEVIRGW EEGVAQMSVG QRAKLTISPD YAYGATGHP GIIPPHATLV FDVELLKLE

UniProtKB: Peptidyl-prolyl cis-trans isomerase FKBP1A

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Macromolecule #4: (2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-ol

MacromoleculeName: (2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-ol
type: ligand / ID: 4 / Number of copies: 1 / Formula: CAU
Molecular weightTheoretical: 298.379 Da
Chemical component information

ChemComp-CAU:
(2S)-1-(9H-Carbazol-4-yloxy)-3-(isopropylamino)propan-2-ol

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Macromolecule #5: 8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN

MacromoleculeName: 8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN / type: ligand / ID: 5 / Number of copies: 1 / Formula: FK5
Molecular weightTheoretical: 804.018 Da
Chemical component information

ChemComp-FK5:
8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN / medication*YM

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2rh1

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 276093
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: NOT APPLICABLE

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