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- EMDB-25143: Structure of positive allosteric modulator-bound active human cal... -

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Basic information

Entry
Database: EMDB / ID: EMD-25143
TitleStructure of positive allosteric modulator-bound active human calcium-sensing receptor
Map data
Sample
  • Complex: Homodimer human calcium sensing receptor
    • Protein or peptide: Isoform 1 of Extracellular calcium-sensing receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CYCLOMETHYLTRYPTOPHAN
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: CALCIUM IONCalcium
  • Ligand: 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine
  • Ligand: CHOLESTEROL
Function / homologyIsoform 1 of Extracellular calcium-sensing receptor
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsPark J / Zuo H / Frangaj A / Fu Z / Yen LY / Zhang Z / Mosyak L / Slavkovich VN / Liu J / Ray KM ...Park J / Zuo H / Frangaj A / Fu Z / Yen LY / Zhang Z / Mosyak L / Slavkovich VN / Liu J / Ray KM / Cao B / Vallese F / Geng Y / Chen S / Grassucci R / Dandey VP / Tan YZ / Eng E / Lee Y / Kloss B / Liu Z / Hendrickson WA / Potter CS / Carragher B / Graziano J / Conigrave AD / Frank J / Clarke OB / Fan QR
Funding support United States, 6 items
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, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sil
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25143.png

Downloads & links

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Map

FileDownload / File: emd_25143.map.gz / Format: CCP4 / Size: 16 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.5
Minimum - Maximum-0.9414485 - 13.26511
Average (Standard dev.)0.059817955 (±0.25118178)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin190189153
Dimensions132135235
Spacing135132235
CellA: 112.049995 Å / B: 109.56 Å / C: 195.05 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z135132235
origin x/y/z0.0000.0000.000
length x/y/z112.050109.560195.050
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS189190153
NC/NR/NS135132235
D min/max/mean-0.94113.2650.060

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Supplemental data

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Sample components

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Entire : Homodimer human calcium sensing receptor

EntireName: Homodimer human calcium sensing receptor
Components
  • Complex: Homodimer human calcium sensing receptor
    • Protein or peptide: Isoform 1 of Extracellular calcium-sensing receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CYCLOMETHYLTRYPTOPHAN
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: CALCIUM IONCalcium
  • Ligand: 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine
  • Ligand: CHOLESTEROL

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Supramolecule #1: Homodimer human calcium sensing receptor

SupramoleculeName: Homodimer human calcium sensing receptor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293 GnTl- / Recombinant plasmid: modified bacMam
Molecular weightTheoretical: 192 KDa

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Macromolecule #1: Isoform 1 of Extracellular calcium-sensing receptor

MacromoleculeName: Isoform 1 of Extracellular calcium-sensing receptor / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.299461 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAFYSCCWVL LALTWHTSAY GPDQRAQKKG DIILGGLFPI HFGVAAKDQD LKSRPESVEC IRYNFRGFRW LQAMIFAIEE INSSPALLP NLTLGYRIFD TCNTVSKALE ATLSFVAQNK IDSLNLDEFC NCSEHIPSTI AVVGATGSGV STAVANLLGL F YIPQVSYA ...String:
MAFYSCCWVL LALTWHTSAY GPDQRAQKKG DIILGGLFPI HFGVAAKDQD LKSRPESVEC IRYNFRGFRW LQAMIFAIEE INSSPALLP NLTLGYRIFD TCNTVSKALE ATLSFVAQNK IDSLNLDEFC NCSEHIPSTI AVVGATGSGV STAVANLLGL F YIPQVSYA SSSRLLSNKN QFKSFLRTIP NDEHQATAMA DIIEYFRWNW VGTIAADDDY GRPGIEKFRE EAEERDICID FS ELISQYS DEEEIQHVVE VIQNSTAKVI VVFSSGPDLE PLIKEIVRRN ITGKIWLASE AWASSSLIAM PQYFHVVGGT IGF ALKAGQ IPGFREFLKK VHPRKSVHNG FAKEFWEETF NCHLQEGAKG PLPVDTFLRG HEESGDRFSN SSTAFRPLCT GDEN ISSVE TPYIDYTHLR ISYNVYLAVY SIAHALQDIY TCLPGRGLFT NGSCADIKKV EAWQVLKHLR HLNFTNNMGE QVTFD ECGD LVGNYSIINW HLSPEDGSIV FKEVGYYNVY AKKGERLFIN EEKILWSGFS REVPFSNCSR DCLAGTRKGI IEGEPT CCF ECVECPDGEY SDETDASACN KCPDDFWSNE NHTSCIAKEI EFLSWTEPFG IALTLFAVLG IFLTAFVLGV FIKFRNT PI VKATNRELSY LLLFSLLCCF SSSLFFIGEP QDWTCRLRQP AFGISFVLCI SCILVKTNRV LLVFEAKIPT SFHRKWWG L NLQFLLVFLC TFMQIVICVI WLYTAPPSSY RNQELEDEII FITCHEGSLM ALGFLIGYTC LLAAICFFFA FKSRKLPEN FNEAKFITFS MLIFFIVWIS FIPAYASTYG KFVSAVEVIA ILAASFGLLA CIFFNKIYII LFKPSRNTIE DYKDDDDK

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #4: CYCLOMETHYLTRYPTOPHAN

MacromoleculeName: CYCLOMETHYLTRYPTOPHAN / type: ligand / ID: 4 / Number of copies: 2 / Formula: TCR
Molecular weightTheoretical: 216.236 Da
Chemical component information

ChemComp-TCR:
CYCLOMETHYLTRYPTOPHAN

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Macromolecule #5: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #7: 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine

MacromoleculeName: 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine
type: ligand / ID: 7 / Number of copies: 2 / Formula: 9IG
Molecular weightTheoretical: 303.826 Da
Chemical component information

ChemComp-9IG:
3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine

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Macromolecule #8: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 8 / Number of copies: 8 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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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

Concentration2.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chlorideSodium chloride
10.0 mMCaCl2Calcium chloride
30.0 uMC12H12N2O2TNCA
30.0 uMC18H22ClNOHClR568 HCl
0.02 %C56H92O25glycol-diosgenin
0.004 %C31H50O4Cholesteryl Hemisuccinate

Details: Solution were made fresh from concentrated, and filtered to avoid microbial contamination.
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum ER / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMax: 100.0 K
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13246 / Average electron dose: 70.35 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 892000
CTF correctionSoftware - Name: cryoSPARC (ver. 2.16)
Startup modelType of model: OTHER / Details: Ab initio (cryoSPARC)
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.16) / Software - details: non-uniform refinement
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.0)
Details: non-uniform refinement and local refinement in cryoSPARC 3.0
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Details: Global(ECD) 2.7A TM 3.4A / Number images used: 130000

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Atomic model buiding 1

Initial model
PDB IDChain

5k5s

chain_id: A, residue_range: 20-607

5k5s

chain_id: B, residue_range: 20-607
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7sil:
Structure of positive allosteric modulator-bound active human calcium-sensing receptor

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