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- EMDB-30301: Cryo-EM structure of the CGP54626-bound human GABA(B) receptor in... -

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Entry
Database: EMDB / ID: EMD-30301
TitleCryo-EM structure of the CGP54626-bound human GABA(B) receptor in inactive state.
Map dataThe focused refinement composite map of the CGP54626-bound GABAB heterodimer.
Sample
  • Complex: The CGP54626-bound GABAB heterodimer
    • Protein or peptide: Gamma-aminobutyric acid type B receptor subunit 1
    • Protein or peptide: Gamma-aminobutyric acid type B receptor subunit 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (R)-(cyclohexylmethyl)[(2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino}-2-hydroxypropyl]phosphinic acid
KeywordsGABAB / Cryo-EM / GPCR / MEMBRANE PROTEIN
Function / homology
Function and homology information


G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion ...G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion / positive regulation of growth hormone secretion / extracellular matrix protein binding / GABA receptor complex / negative regulation of adenylate cyclase activity / positive regulation of glutamate secretion / Class C/3 (Metabotropic glutamate/pheromone receptors) / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / negative regulation of synaptic transmission / axolemma / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / dendritic shaft / response to nicotine / mitochondrial membrane / Schaffer collateral - CA1 synapse / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / osteoblast differentiation / transmembrane signaling receptor activity / synaptic vesicle / presynaptic membrane / G alpha (i) signalling events / chemical synaptic transmission / postsynaptic membrane / response to ethanol / dendritic spine / neuron projection / protein heterodimerization activity / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / neuronal cell body / glutamatergic synapse / endoplasmic reticulum membrane / extracellular space / plasma membrane / cytoplasm
Similarity search - Function
GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / G-protein coupled receptors family 3 profile. / GPCR family 3, C-terminal ...GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / G-protein coupled receptors family 3 profile. / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
Gamma-aminobutyric acid type B receptor subunit 2 / Gamma-aminobutyric acid type B receptor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMao C / Shen C
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81922071 China
Ministry of Science and Technology (MoST, China)2018YFA0507003 China
CitationJournal: Cell Res / Year: 2020
Title: Cryo-EM structures of inactive and active GABA receptor.
Authors: Chunyou Mao / Cangsong Shen / Chuntao Li / Dan-Dan Shen / Chanjuan Xu / Shenglan Zhang / Rui Zhou / Qingya Shen / Li-Nan Chen / Zhinong Jiang / Jianfeng Liu / Yan Zhang /
Abstract: Metabotropic GABA G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is ...Metabotropic GABA G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABA receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of G protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of G. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABA receptor.
History
DepositionMay 26, 2020-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-7c7s
  • Surface level: 0.04
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30301.png

Downloads & links

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Map

FileDownload / File: emd_30301.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe focused refinement composite map of the CGP54626-bound GABAB heterodimer.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 320 pix.
= 324.48 Å		1.01 Å/pix.
x 320 pix.
= 324.48 Å		1.01 Å/pix.
x 320 pix.
= 324.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.014 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.038844854 - 0.33748716
Average (Standard dev.)0.00021128019 (±0.0043344493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 324.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z324.480324.480324.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0390.3370.000

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Supplemental data

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Additional map: The overall refinement map of the CGP54626-bound GABAB heterodimer.

Fileemd_30301_additional.map
AnnotationThe overall refinement map of the CGP54626-bound GABAB heterodimer.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The CGP54626-bound GABAB heterodimer

EntireName: The CGP54626-bound GABAB heterodimer
Components
  • Complex: The CGP54626-bound GABAB heterodimer
    • Protein or peptide: Gamma-aminobutyric acid type B receptor subunit 1
    • Protein or peptide: Gamma-aminobutyric acid type B receptor subunit 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (R)-(cyclohexylmethyl)[(2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino}-2-hydroxypropyl]phosphinic acid

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Supramolecule #1: The CGP54626-bound GABAB heterodimer

SupramoleculeName: The CGP54626-bound GABAB heterodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Gamma-aminobutyric acid type B receptor subunit 1

MacromoleculeName: Gamma-aminobutyric acid type B receptor subunit 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 105.976297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKTIIALSYI FCLVFAPGAG GAQTPNATSE GCQIIHPPWE GGIRYRGLTR DQVKAINFLP VDYEIEYVCR GEREVVGPKV RKCLANGSW TDMDTPSRCV RICSKSYLTL ENGKVFLTGG DLPALDGARV DFRCDPDFHL VGSSRSICSQ GQWSTPKPHC Q VNRTPHSE ...String:
MKTIIALSYI FCLVFAPGAG GAQTPNATSE GCQIIHPPWE GGIRYRGLTR DQVKAINFLP VDYEIEYVCR GEREVVGPKV RKCLANGSW TDMDTPSRCV RICSKSYLTL ENGKVFLTGG DLPALDGARV DFRCDPDFHL VGSSRSICSQ GQWSTPKPHC Q VNRTPHSE RRAVYIGALF PMSGGWPGGQ ACQPAVEMAL EDVNSRRDIL PDYELKLIHH DSKCDPGQAT KYLYELLYND PI KIILMPG CSSVSTLVAE AARMWNLIVL SYGSSSPALS NRQRFPTFFR THPSATLHNP TRVKLFEKWG WKKIATIQQT TEV FTSTLD DLEERVKEAG IEITFRQSFF SDPAVPVKNL KRQDARIIVG LFYETEARKV FCEVYKERLF GKKYVWFLIG WYAD NWFKI YDPSINCTVD EMTEAVEGHI TTEIVMLNPA NTRSISNMTS QEFVEKLTKR LKRHPEETGG FQEAPLAYDA IWALA LALN KTSGGGGRSG VRLEDFNYNN QTITDQIYRA MNSSSFEGVS GHVVFDASGS RMAWTLIEQL QGGSYKKIGY YDSTKD DLS WSKTDKWIGG SPPADQTLVI KTFRFLSQKL FISVSVLSSL GIVLAVVCLS FNIYNSHVRY IQNSQPNLNN LTAVGCS LA LAAVFPLGLD GYHIGRNQFP FVCQARLWLL GLGFSLGYGS MFTKIWWVHT VFTKKEEKKE WRKTLEPWKL YATVGLLV G MDVLTLAIWQ IVDPLHRTIE TFAKEEPKED IDVSILPQLE HCSSRKMNTW LGIFYGYKGL LLLLGIFLAY ETKSVSTEK INDHRAVGMA IYNVAVLCLI TAPVTMILSS QQDAAFAFAS LAIVFSSYIT LVVLFVPKMR RLITRGEWQS EAQDTMKTGS STNNNEEEK SRLLEKENRE LEKIIAEKEE RVSELRHQLQ SRLEVLFQGP HHHHHHHH

UniProtKB: Gamma-aminobutyric acid type B receptor subunit 1

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Macromolecule #2: Gamma-aminobutyric acid type B receptor subunit 2

MacromoleculeName: Gamma-aminobutyric acid type B receptor subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 91.359445 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKTIIALSYI FCLVFADYKD DDDKGSGGSG WARGAPRPPP SSPPLSIMGL MPLTKEVAKG SIGRGVLPAV ELAIEQIRNE SLLRPYFLD LRLYDTECDN AKGLKAFYDA IKYGPNHLMV FGGVCPSVTS IIAESLQGWN LVQLSFAATT PVLADKKKYP Y FFRTVPSD ...String:
MKTIIALSYI FCLVFADYKD DDDKGSGGSG WARGAPRPPP SSPPLSIMGL MPLTKEVAKG SIGRGVLPAV ELAIEQIRNE SLLRPYFLD LRLYDTECDN AKGLKAFYDA IKYGPNHLMV FGGVCPSVTS IIAESLQGWN LVQLSFAATT PVLADKKKYP Y FFRTVPSD NAVNPAILKL LKHYQWKRVG TLTQDVQRFS EVRNDLTGVL YGEDIEISDT ESFSNDPCTS VKKLKGNDVR II LGQFDQN MAAKVFCCAY EENMYGSKYQ WIIPGWYEPS WWEQVHTEAN SSRCLRKNLL AAMEGYIGVD FEPLSSKQIK TIS GKTPQQ YEREYNNKRS GVGPSKFHGY AYDGIWVIAK TLQRAMETLH ASSRHQRIQD FNYTDHTLGR IILNAMNETN FFGV TGQVV FRNGERMGTI KFTQFQDSRE VKVGEYNAVA DTLEIINDTI RFQGSEPPKD KTIILEQLRK ISLPLYSILS ALTIL GMIM ASAFLFFNIK NRNQKLIKMS SPYMNNLIIL GGMLSYASIF LFGLDGSFVS EKTFETLCTV RTWILTVGYT TAFGAM FAK TWRVHAIFKN VKMKKKIIKD QKLLVIVGGM LLIDLCILIC WQAVDPLRRT VEKYSMEPDP AGRDISIRPL LEHCENT HM TIWLGIVYAY KGLLMLFGCF LAWETRNVSI PALNDSKYIG MSVYNVGIMC IIGAAVSFLT RDQPNVQFCI VALVIIFC S TITLCLVFVP KLITLRTNPD AATQNRRFQF TQNQKKEDSK TSTSVTSVNQ ASTSRLEGLQ SENHRLRMKI TELDKDLEE VTMQLQDT

UniProtKB: Gamma-aminobutyric acid type B receptor subunit 2

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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: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: (R)-(cyclohexylmethyl)[(2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]...

MacromoleculeName: (R)-(cyclohexylmethyl)[(2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino}-2-hydroxypropyl]phosphinic acid
type: ligand / ID: 4 / Number of copies: 1 / Formula: 2BV
Molecular weightTheoretical: 408.3 Da
Chemical component information

ChemComp-2BV:
(R)-(cyclohexylmethyl)[(2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino}-2-hydroxypropyl]phosphinic acid

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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 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormula
50.0 mMHEPES
150.0 mMNaCl
2.0 mMMgCl2
0.002 (w/v)%LMNG
0.0004 (w/v)%CHS
10.0 uMCGP54626
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number real images: 4740 / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49310 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 29000
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: 2969413
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta2) / Number images used: 374589
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

4mr7


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7c7s:
Cryo-EM structure of the CGP54626-bound human GABA(B) receptor in inactive state.

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