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- PDB-7sil: Structure of positive allosteric modulator-bound active human cal... -

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Basic information

Entry
Database: PDB / ID: 7sil
TitleStructure of positive allosteric modulator-bound active 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-9IG / CHOLESTEROL / PHOSPHATE ION / CYCLOMETHYLTRYPTOPHAN / Isoform 1 of Extracellular calcium-sensing receptor
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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

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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)206,26834
Polymers198,5992
Non-polymers7,66932
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17430 Å2
ΔGint-90 kcal/mol
Surface area67530 Å2

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Components

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Protein , 1 types, 2 molecules AB

#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

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Sugars , 2 types, 10 molecules

#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 22 molecules

#4: Chemical ChemComp-TCR / CYCLOMETHYLTRYPTOPHAN


Type: L-peptide linking / Mass: 216.236 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C12H12N2O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-9IG / 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine


Mass: 303.826 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H22ClNO / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C27H46O

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Details

Has ligand of interestY

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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: 7.5
Details: Solution were made fresh from concentrated, and filtered to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2100 mMSodium chlorideNaClSodium chloride1
310 mMCalcium chlorideCaCl21
430 uMTNCAC12H12N2O21
530 uMR568 HClC18H22ClNOHCl1
60.02 %glycol-diosgeninC56H92O251
70.004 %Cholesteryl HemisuccinateC31H50O41
SpecimenConc.: 2.6 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 4 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 FIELDBright-field microscopy / 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 recordingElectron dose: 70.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13246
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / 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
12RELION3.1classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 892000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130000 / Algorithm: FOURIER SPACE / Details: Global(ECD) 2.7A TM 3.4A / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15K5S

5k5s

A120-607
25K5S

5k5s

B120-607
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313256
ELECTRON MICROSCOPYf_angle_d0.53618032
ELECTRON MICROSCOPYf_dihedral_angle_d6.3131844
ELECTRON MICROSCOPYf_chiral_restr0.0422066
ELECTRON MICROSCOPYf_plane_restr0.0042212

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