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- EMDB-43907: Local refinement map of G protein in nanodisc-reconstituted human... -

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Basic information

Entry
Database: EMDB / ID: EMD-43907
TitleLocal refinement map of G protein in nanodisc-reconstituted human CaSR-Gi3 complex
Map dataLocal refinement map of G protein in nanodisc-reconstituted human CaSR-Gi3 complex
Sample
  • Complex: Human CaSR in complex with Gi3
KeywordsCalcium-sensing receptor / G-protein-coupled receptor / G protein / signal transduction / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZuo H / Park J / Frangaj A / Ye J / Lu G / Manning JJ / Asher WB / Lu Z / Hu G / Wang L ...Zuo H / Park J / Frangaj A / Ye J / Lu G / Manning JJ / Asher WB / Lu Z / Hu G / Wang L / Mendez J / Eng E / Zhang Z / Lin X / Grasucci R / Hendrickson WA / Clarke OB / Javitch JA / Conigrave AD / Fan QR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141871 United States
CitationJournal: Nature / Year: 2024
Title: Promiscuous G-protein activation by the calcium-sensing receptor.
Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / ...Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / Xin Lin / Robert Grassucci / Wayne A Hendrickson / Oliver B Clarke / Jonathan A Javitch / Arthur D Conigrave / Qing R Fan /
Abstract: The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca ...The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca balance. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes. We determined structures of CaSR in complex with G proteins from three different subfamilies: G, G and G. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines G and G versus G selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.
History
DepositionMar 4, 2024-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43907.png

Downloads & links

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Map

FileDownload / File: emd_43907.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement map of G protein in nanodisc-reconstituted human CaSR-Gi3 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 512 pix.
= 432.282 Å		0.84 Å/pix.
x 512 pix.
= 432.282 Å		0.84 Å/pix.
x 512 pix.
= 432.282 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8443 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.18915825 - 0.35771698
Average (Standard dev.)-0.000013425151 (±0.0057662767)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 432.2816 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: G protein local refinement half map A of CaSR-Gi3

Fileemd_43907_half_map_1.map
AnnotationG protein local refinement half map A of CaSR-Gi3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: G protein local refinement half map B of CaSR-Gi3

Fileemd_43907_half_map_2.map
AnnotationG protein local refinement half map B of CaSR-Gi3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human CaSR in complex with Gi3

EntireName: Human CaSR in complex with Gi3
Components
  • Complex: Human CaSR in complex with Gi3

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Supramolecule #1: Human CaSR in complex with Gi3

SupramoleculeName: Human CaSR in complex with Gi3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 288 KDa

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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

Concentration3.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chloride
2.0 mMMgCl2Magnesium chloride
10.0 mMCaCl2Calcium chloride
20.0 uMC12H12N2O2TNCA
20.0 uMC18H22ClNOHClR568 HCl
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was blotted for 6s before plunge-frozen..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMax: 100.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
SoftwareName: Leginon (ver. 3.6)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 16504 / Average exposure time: 2.5 sec. / Average electron dose: 70.14 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2792317
Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 55985
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7sil

chain_id: Q, source_name: PDB, initial_model_type: experimental model

7sil

chain_id: R, source_name: PDB, initial_model_type: experimental model

7s8m

chain_id: A, source_name: PDB, initial_model_type: experimental model

7s8m

chain_id: B, source_name: PDB, initial_model_type: experimental model

7s8m

chain_id: G, source_name: PDB, initial_model_type: experimental model
SoftwareName: Coot (ver. 0.9.8.1)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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