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- PDB-9chv: cryo-EM structure of calcineurin-fused beta2 adrenergic receptor ... -

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Basic information

Entry
Database: PDB / ID: 9chv
Titlecryo-EM structure of calcineurin-fused beta2 adrenergic receptor in apo state
Components
  • Beta-2 adrenergic receptor,Calcineurin subunit B type 1
  • Peptidyl-prolyl cis-trans isomerase FKBP1A
  • Protein phosphatase 3 catalytic subunit alpha
KeywordsMEMBRANE PROTEIN/HYDROLASE/ISOMERASE / GPCR / 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 saliva secretion ...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 saliva secretion / negative regulation of dendrite morphogenesis / calcineurin complex / positive regulation of connective tissue replacement / positive regulation of calcium ion import across plasma membrane / slit diaphragm / positive regulation of cardiac muscle hypertrophy in response to stress / FCERI mediated Ca+2 mobilization / Ca2+ pathway / protein serine/threonine phosphatase complex / 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 / type I transforming growth factor beta receptor binding / myelination in peripheral nervous system / negative regulation of activin receptor signaling pathway / transition between fast and slow fiber / heart trabecula formation / positive regulation of osteoclast differentiation / I-SMAD binding / cardiac muscle hypertrophy in response to stress / positive regulation of mini excitatory postsynaptic potential / beta2-adrenergic receptor activity / AMPA selective glutamate receptor signaling pathway / signaling receptor inhibitor activity / regulation of synaptic vesicle cycle / regulation of amyloid precursor protein catabolic process / terminal cisterna / ryanodine receptor complex / norepinephrine binding / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / Adrenoceptors / 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 G protein-coupled receptor signaling pathway / negative regulation of multicellular organism growth / CLEC7A (Dectin-1) induces NFAT activation / ventricular cardiac muscle tissue morphogenesis / adrenergic receptor signaling pathway / branching involved in blood vessel morphogenesis / 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 / endosome to lysosome transport / myosin phosphatase activity / protein-serine/threonine phosphatase / FK506 binding / regulation of postsynaptic neurotransmitter receptor internalization / parallel fiber to Purkinje cell synapse / diet induced thermogenesis / positive regulation of cardiac muscle hypertrophy / neuronal dense core vesicle / positive regulation of activated T cell proliferation / positive regulation of endocytosis / TGF-beta receptor signaling activates SMADs / mTORC1-mediated signalling / positive regulation of cAMP/PKA signal transduction / phosphoprotein phosphatase activity / Calcineurin activates NFAT / adenylate cyclase binding / 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 / regulation of immune response / bone resorption / positive regulation of bone mineralization / potassium channel regulator activity / postsynaptic modulation of chemical synaptic transmission / positive regulation of osteoblast differentiation / multicellular organismal response to stress
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 / FKBP-type peptidyl-prolyl cis-trans isomerase domain / 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
8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsXu, J. / Chen, G. / Du, Y. / Kobilka, B.K.
Funding support1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Database references / Structure summary
Category: audit_author / citation ...audit_author / citation / citation_author / em_admin / em_author_list / pdbx_contact_author
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-2 adrenergic receptor,Calcineurin subunit B type 1
B: Protein phosphatase 3 catalytic subunit alpha
C: Peptidyl-prolyl cis-trans isomerase FKBP1A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,7094
Polymers105,9053
Non-polymers8041
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 51309.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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
Source: (gene. exp.) Homo sapiens (human) / Gene: ADRB2, ADRB2R, B2AR, PPP3R1, CNA2, CNB / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P07550, UniProt: P63098
#2: Protein Protein phosphatase 3 catalytic subunit alpha / CAM-PRP catalytic subunit / Calcineurin A alpha / Calmodulin-dependent calcineurin A subunit alpha ...CAM-PRP catalytic subunit / Calcineurin A alpha / Calmodulin-dependent calcineurin A subunit alpha isoform / CNA alpha / Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform


Mass: 42627.785 Da / Num. of mol.: 1 / Fragment: residues 1-370
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ppp3ca, Calna / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P63328, protein-serine/threonine phosphatase
#3: Protein Peptidyl-prolyl cis-trans isomerase FKBP1A / PPIase FKBP1A / 12 kDa FK506-binding protein / FKBP-12 / Calstabin-1 / FK506-binding protein 1A / ...PPIase FKBP1A / 12 kDa FK506-binding protein / FKBP-12 / Calstabin-1 / FK506-binding protein 1A / FKBP-1A / Immunophilin FKBP12 / Rotamase


Mass: 11967.705 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FKBP1A, FKBP1, FKBP12
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P62942, peptidylprolyl isomerase
#4: Chemical ChemComp-FK5 / 8-DEETHYL-8-[BUT-3-ENYL]-ASCOMYCIN / K506


Mass: 804.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C44H69NO12 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1calcineurin fused beta2AR in complex with FKBP12COMPLEX#1-#30MULTIPLE SOURCES
2calcineurin fused beta2ARCOMPLEX#11RECOMBINANT
3Protein phosphatase 3 catalytic subunit alphaCOMPLEX#21RECOMBINANT
4FKBP1ACOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Mus musculus (house mouse)10090
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Spodoptera frugiperda (fall armyworm)7108
44Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18_3861: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 279590 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047093
ELECTRON MICROSCOPYf_angle_d0.6429632
ELECTRON MICROSCOPYf_dihedral_angle_d14.224997
ELECTRON MICROSCOPYf_chiral_restr0.0421093
ELECTRON MICROSCOPYf_plane_restr0.0041227

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