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- PDB-7sin: Structure of negative allosteric modulator-bound inactive human c... -

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Basic information

Entry
Database: PDB / ID: 7sin
TitleStructure of negative allosteric modulator-bound inactive human calcium-sensing receptor
ComponentsIsoform 1 of Extracellular calcium-sensing receptor
KeywordsMEMBRANE PROTEIN / calcium-sensing receptor / cryo-EM structure / allosteric modulation / activation mechanism / symmetry
Function / homologyChem-YP1 / Isoform 1 of Extracellular calcium-sensing receptor
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsPark, J. / Zuo, H. / Frangaj, A. / Fu, Z. / Yen, L.Y. / Zhang, Z. / Mosyak, L. / Slavkovich, V.N. / Liu, J. / Ray, K.M. ...Park, J. / Zuo, H. / Frangaj, A. / Fu, Z. / Yen, L.Y. / Zhang, Z. / Mosyak, L. / Slavkovich, V.N. / Liu, J. / Ray, K.M. / Cao, B. / Vallese, F. / Geng, Y. / Chen, S. / Grassucci, R. / Dandey, V.P. / Tan, Y.Z. / Eng, E. / Lee, Y. / Kloss, B. / Liu, Z. / Hendrickson, W.A. / Potter, C.S. / Carragher, B. / Graziano, J. / Conigrave, A.D. / Frank, J. / Clarke, O.B. / Fan, Q.R.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141871 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM29169 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM116799 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107462 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Symmetric activation and modulation of the human calcium-sensing receptor.
Authors: Jinseo Park / Hao Zuo / Aurel Frangaj / Ziao Fu / Laura Y Yen / Zhening Zhang / Lidia Mosyak / Vesna N Slavkovich / Jonathan Liu / Kimberly M Ray / Baohua Cao / Francesca Vallese / Yong Geng ...Authors: Jinseo Park / Hao Zuo / Aurel Frangaj / Ziao Fu / Laura Y Yen / Zhening Zhang / Lidia Mosyak / Vesna N Slavkovich / Jonathan Liu / Kimberly M Ray / Baohua Cao / Francesca Vallese / Yong Geng / Shaoxia Chen / Robert Grassucci / Venkata P Dandey / Yong Zi Tan / Edward Eng / Yeji Lee / Brian Kloss / Zheng Liu / Wayne A Hendrickson / Clinton S Potter / Bridget Carragher / Joseph Graziano / Arthur D Conigrave / Joachim Frank / Oliver B Clarke / Qing R Fan /
Abstract: The human extracellular calcium-sensing (CaS) receptor controls plasma Ca levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS ...The human extracellular calcium-sensing (CaS) receptor controls plasma Ca levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.
History
DepositionOct 14, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Isoform 1 of Extracellular calcium-sensing receptor
B: Isoform 1 of Extracellular calcium-sensing receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,4174
Polymers198,5992
Non-polymers8182
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4380 Å2
ΔGint-31 kcal/mol
Surface area73340 Å2

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Components

#1: Protein Isoform 1 of Extracellular calcium-sensing receptor / CaR / CaSR / hCasR / Parathyroid cell calcium-sensing receptor 1 / PCaR1


Mass: 99299.461 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CASR, GPRC2A, PCAR1 / Cell line (production host): HEK293 GnTl- / Production host: Homo sapiens (human) / References: UniProt: P41180-1
#2: Chemical ChemComp-YP1 / 2-chloro-6-[(2R)-2-hydroxy-3-{[2-methyl-1-(naphthalen-2-yl)propan-2-yl]amino}propoxy]benzonitrile


Mass: 408.921 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H25ClN2O2 / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: Homodimer human calcium sensing receptor / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.192 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293 GnTl- / Plasmid: modified bacMam
Buffer solutionpH: 6.8
Details: Solution were made fresh from concentrated, and filtered to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2300 mMSodium chlorideNaCl1
30.02 %glycol-diosgeninC56H92O251
40.004 %Cholesteryl HemisuccinateC31H50O41
520 mMLithium SulfateLi2SO41
620 uMNPS-2143C24H25ClN2O21
SpecimenConc.: 1.96 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 6 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 58.63 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15464
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC2.16particle selection
2Leginonimage acquisition
4cryoSPARC2.16CTF correction
7Coot0.9.1model fitting
9PHENIX1.14model refinement
10cryoSPARC2.16initial Euler assignmentnon-uniform refinement
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1241782
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161574 / Algorithm: FOURIER SPACE / Details: Local ECD 4.0A Local TM 4.2A / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 5K5S / Initial refinement model-ID: 1 / Pdb chain residue range: 20-607 / PDB-ID: 5K5S

5k5s

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212986
ELECTRON MICROSCOPYf_angle_d0.51517624
ELECTRON MICROSCOPYf_dihedral_angle_d16.191707
ELECTRON MICROSCOPYf_chiral_restr0.0411958
ELECTRON MICROSCOPYf_plane_restr0.0042236

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