EMDB-50275: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R ...

Basic information

Entry

Database: EMDB / ID: EMD-50275

• Title: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3)
• Map data: Unsharpened map from Refine3D

Sample

• Complex: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3)
  • Protein or peptide: x 6 types
• Ligand: x 8 types

• Keywords: GABA / Neurotransmission / Ternary complex / Inhibitory postsynapse / MEMBRANE PROTEIN

Function / homology

Function:

regulation of inhibitory synapse assembly / symmetric, GABA-ergic, inhibitory synapse / jump response / neurotransmitter-gated ion channel clustering / positive regulation of t-SNARE clustering / gephyrin clustering involved in postsynaptic density assembly / terminal button organization / postsynaptic density protein 95 clustering / positive regulation of synaptic vesicle clustering / postsynaptic membrane assembly / gamma-aminobutyric acid receptor clustering / cell-cell junction maintenance / presynaptic membrane assembly / postsynaptic specialization / thigmotaxis / ribbon synapse / neuron cell-cell adhesion / insulin metabolic process / regulation of respiratory gaseous exchange by nervous system process / neurexin family protein binding / benzodiazepine receptor activity / inhibitory synapse / presynapse assembly / GABA receptor binding / GABA receptor complex / protein localization to synapse / dopaminergic synapse / glycinergic synapse / regulation of AMPA receptor activity / positive regulation of inhibitory postsynaptic potential / cellular response to histamine / GABA receptor activation / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / positive regulation of synapse assembly / protein localization to cell surface / Neurexins and neuroligins / positive regulation of dendritic spine development / neurotransmitter receptor localization to postsynaptic specialization membrane / positive regulation of protein localization to synapse / postsynaptic specialization membrane / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / chloride channel activity / social behavior / adult behavior / locomotory exploration behavior / roof of mouth development / Signaling by ERBB4 / neuromuscular process controlling balance / excitatory synapse / positive regulation of excitatory postsynaptic potential / regulation of presynapse assembly / chloride channel complex / synapse assembly / cell adhesion molecule binding / sensory perception of pain / dendrite membrane / cytoplasmic vesicle membrane / positive regulation of synaptic transmission, glutamatergic / chloride transmembrane transport / dendritic shaft / post-embryonic development / positive regulation of synaptic transmission, GABAergic / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / GABA-ergic synapse / sensory perception of sound / synapse organization / modulation of chemical synaptic transmission / positive regulation of insulin secretion / cell-cell adhesion / presynaptic membrane / dendritic spine / postsynaptic membrane / postsynapse / axon / positive regulation of cell population proliferation / synapse / dendrite / cell surface / signal transduction / identical protein binding / membrane / plasma membrane

Domain/homology:

Lipoma HMGIC fusion partner-like protein / Lipoma HMGIC fusion partner-like protein / Neuroligin / : / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / : / Gamma-aminobutyric-acid A receptor, beta subunit / Carboxylesterase type B, conserved site / Carboxylesterases type-B signature 2. / Carboxylesterase, type B / Carboxylesterase family / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain / Alpha/Beta hydrolase fold

Component:

Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-3 / LHFPL tetraspan subfamily member 4 protein / Neuroligin-2

• Biological species: Homo sapiens (human) / Lama glama (llama)
• Method: single particle reconstruction / cryo EM / Resolution: 3.5 Å
• Authors: Kasaragod VB / Aricescu AR

Funding support

United Kingdom, European Union, United States, 7 items

Organization	Grant number	Country
Medical Research Council (MRC, United Kingdom)	MC_UP_1201/15	United Kingdom
European Molecular Biology Organization (EMBO)	ALTF137-2019	European Union
H2020 Marie Curie Actions of the European Commission	GABAARComp-897707	European Union
Medical Research Council (MRC, United Kingdom)	MR/L009609/1	United Kingdom
Medical Research Council (MRC, United Kingdom)	MC_EX_MR/T046279/1	United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	1R01-GM135550	United States
National Science Foundation (NSF, United States)	NeuroNex 2014862	United States

• Citation: Journal: To Be Published; Title: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody in a desensitised state (StateD3); Authors: Kasaragod VB / Aricescu AR

History

Deposition	May 10, 2024	-
Header (metadata) release	Jul 2, 2025	-
Map release	Jul 2, 2025	-
Update	Jul 2, 2025	-
Current status	Jul 2, 2025	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_50275.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Unsharpened map from Refine3D
• Voxel size: X=Y=Z: 0.79297 Å

Density

Contour Level	By AUTHOR: 0.01
Minimum - Maximum	-0.022358963 - 0.04790236
Average (Standard dev.)	0.00028118346 (±0.0027381363)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 296 296 296 Spacing 296 296 296
Cell	A=B=C: 234.71912 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_50275_msk_1.map

Additional map: Postprocessed map

• File: emd_50275_additional_1.map
• Annotation: Postprocessed map

Half map: One of the halfmaps

• File: emd_50275_half_map_1.map
• Annotation: One of the halfmaps

Half map: One of the halfmaps

• File: emd_50275_half_map_2.map
• Annotation: One of the halfmaps

Sample components

Entire : CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R ...

• Entire: Name: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3)

Components

• Complex: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3)
  • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
  • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3
  • Protein or peptide: Isoform 2 of Gamma-aminobutyric acid receptor subunit gamma-2
  • Protein or peptide: Neuroligin-2
  • Protein or peptide: LHFPL tetraspan subfamily member 4 protein
  • Protein or peptide: Megabody38
• Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
• Ligand: GAMMA-AMINO-BUTANOIC ACID
• Ligand: CHLORIDE ION
• Ligand: HEXANE
• Ligand: DECANE
• Ligand: PALMITIC ACID
• Ligand: CHOLESTEROL
• Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

Supramolecule #1: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R ...

• Supramolecule: Name: CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3); type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 300 KDa

Macromolecule #1: Gamma-aminobutyric acid receptor subunit alpha-1

• Macromolecule: Name: Gamma-aminobutyric acid receptor subunit alpha-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 47.053117 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

DNTTVFTRIL DRLLDGYDNR LRPGLGERVT EVKTDIFVTS FGPVSDHDME YTIDVFFRQS WKDERLKFKG PMTVLRLNNL MASKIWTPD TFFHNGKKSV AHNMTMPNKL LRITEDGTLL YTMRLTVRAE CPMHLEDFPM DAHACPLKFG SYAYTRAEVV Y EWTREPAR SVVVAEDGSR LNQYDLLGQT VDSGIVQSST GEYVVMTTHF HLKRKIGYFV IQTYLPCIMT VILSQVSFWL NR ESVPART VFGVTTVLTM TTLSISARNS LPKVAYATAM DWFIAVCYAF VFSALIEFAT VNYFTKRGYA WDGKSVVPEK PKK VKDPLI KKNNTYAPTA TSYTPNLARG DPGLATIAKS ATIEPKEVKP ETKPPEPKKT FNSVSKIDRL SRIAFPLLFG IFNL VYWAT YLNREPQL

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-1

Macromolecule #2: Gamma-aminobutyric acid receptor subunit beta-3

• Macromolecule: Name: Gamma-aminobutyric acid receptor subunit beta-3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 50.904527 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

GNMSFVKETV DKLLKGYDIR LRPDFGGPPV CVGMNIDIAS IDMVSEVNMD YTLTMYFQQY WRDKRLAYSG IPLNLTLDNR VADQLWVPD TYFLNDKKSF VHGVTVKNRM IRLHPDGTVL YGLRITTTAA CMMDLRRYPL DEQNCTLEIE SYGYTTDDIE F YWRGGDKA VTGVERIELP QFSIVEHRLV SRNVVFATGA YPRLSLSFRL KRNIGYFILQ TYMPSILITI LSWVSFWINY DA SAARVAL GITTVLTMTT INTHLRETLP KIPYVKAIDM YLMGCFVFVF LALLEYAFVN YIFFGRGPQR QKKLAEKTAK AKN DRSKSE SNRVDAHGNI LLTSLEVHNE MNEVSGGIGD TRNSAISFDN SGIQYRKQSM PREGHGRFLG DRSLPHKKTH LRRR SSQLK IKIPDLTDVN AIDRWSRIVF PFTFSLFNLV YWLYYV

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-3

Macromolecule #3: Isoform 2 of Gamma-aminobutyric acid receptor subunit gamma-2

• Macromolecule: Name: Isoform 2 of Gamma-aminobutyric acid receptor subunit gamma-2; type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 47.870398 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

GDVTVILNNL LEGYDNKLRP DIGVKPTLIH TDMYVNSIGP VNAINMEYTI DIFFAQTWYD RRLKFNSTIK VLRLNSNMVG KIWIPDTFF RNSKKADAHW ITTPNRMLRI WNDGRVLYTL RLTIDAECQL QLHNFPMDEH SCPLEFSSYG YPREEIVYQW K RSSVEVGD TRSWRLYQFS FVGLRNTTEV VKTTSGDYVV MSVYFDLSRR MGYFTIQTYI PCTLIVVLSW VSFWINKDAV PA RTSLGIT TVLTMTTLST IARKSLPKVS YVTAMDLFVS VCFIFVFSAL VEYGTLHYFV SNRKPSKDKD KKKKNPAPTI DIR PRSATI QMNNATHLQE RDEEYGYECL DGKDCASFF(P1L) (P1L)FED(P1L)RTGAW RHGRIHIRIA KMDSYARIFF PT AFCLFNL VYWVSYLYLG

UniProtKB: Gamma-aminobutyric acid receptor subunit gamma-2

Macromolecule #4: Neuroligin-2

• Macromolecule: Name: Neuroligin-2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 3.614107 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

DSRDYSTELS VTVAVGASLL FLNILAFAAL YYK

UniProtKB: Neuroligin-2

Macromolecule #5: LHFPL tetraspan subfamily member 4 protein

• Macromolecule: Name: LHFPL tetraspan subfamily member 4 protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 21.366189 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

YHEHYMRNSR AIGVLWAIFT ICFAIINVVV FIQPYWVGDS VSTPKPGYFG LFHYCVGSGL AGRELTCRGS FTDFSTIPSS AFKAAAFFV LLSMVLILGC ITCFSLFFFC NTATVYKICA WMQLLAALCL VLGCMIFPDG WDAETIRDMC GAKTGKYSLG D CSVRWAYI LAIIGILNAL ILSFLAFVLG NRQTD

UniProtKB: LHFPL tetraspan subfamily member 4 protein

Macromolecule #6: Megabody38

• Macromolecule: Name: Megabody38 / type: protein_or_peptide / ID: 6 ; Details: This megabody is a chimeric protein designed and constructed based on a circular permutation of a nanobody (raised against GABA(A) receptor subunits, in Llama) and Helicobacter pylori protein, HopQ (Uniprot Id: B5Z8H1).; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Lama glama (llama)
• Molecular weight: Theoretical: 57.784301 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

QVQLQESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK CDASAISSAN MTMQNQKNNW GNGCAGVEET QSLLKTSAAD FNNQTPQINQ AQNLANTLIQ ELGNNPFRAS GG GSGGGGS GKLSDTYEQL SRLLTNDNGT NSKTSAQAIN QAVNNLNERA KTLAGGTTNS PAYQATLLAL RSVLGLWNSM GYA VICGGY TKSPGENNQK DFHYTDENGN GTTINCGGST NSNGTHSYNG TNTLKADKNV SLSIEQYEKI HEAYQILSKA LKQA GLAPL NSKGEKLEAH VTTSKYGSLR VSCAASGRTF TTYIMAWFRQ APGKEREFLA AMDQGRIQYY GDSVRGRFTI SRDYA KNSV DLQLDGLRPE DTAVYYCAAG AGFWGLRTAS SYHYWGQGTQ VTVSSHHHHH HEPEA

Macromolecule #12: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

• Macromolecule: Name: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate; type: ligand / ID: 12 / Number of copies: 2 / Formula: PIO
• Molecular weight: Theoretical: 746.566 Da

Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

Macromolecule #13: GAMMA-AMINO-BUTANOIC ACID

• Macromolecule: Name: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 13 / Number of copies: 2 / Formula: ABU
• Molecular weight: Theoretical: 103.12 Da

Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC ACID

Macromolecule #14: CHLORIDE ION

• Macromolecule: Name: CHLORIDE ION / type: ligand / ID: 14 / Number of copies: 3 / Formula: CL
• Molecular weight: Theoretical: 35.453 Da

Macromolecule #15: HEXANE

• Macromolecule: Name: HEXANE / type: ligand / ID: 15 / Number of copies: 7 / Formula: HEX
• Molecular weight: Theoretical: 86.175 Da

Chemical component information

ChemComp-HEX:
HEXANE

Macromolecule #16: DECANE

• Macromolecule: Name: DECANE / type: ligand / ID: 16 / Number of copies: 1 / Formula: D10
• Molecular weight: Theoretical: 142.282 Da

Chemical component information

ChemComp-D10:
DECANE

Macromolecule #17: PALMITIC ACID

• Macromolecule: Name: PALMITIC ACID / type: ligand / ID: 17 / Number of copies: 1 / Formula: PLM
• Molecular weight: Theoretical: 256.424 Da

Chemical component information

ChemComp-PLM:
PALMITIC ACID

Macromolecule #18: CHOLESTEROL

• Macromolecule: Name: CHOLESTEROL / type: ligand / ID: 18 / Number of copies: 1 / Formula: CLR
• Molecular weight: Theoretical: 386.654 Da

Chemical component information

ChemComp-CLR:
CHOLESTEROL

Macromolecule #19: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

• Macromolecule: Name: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate; type: ligand / ID: 19 / Number of copies: 2 / Formula: PGW
• Molecular weight: Theoretical: 749.007 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1 mg/mL

Buffer

pH: 7.4
Component:

Concentration	Formula	Name
15.0 mM	HEPES	4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mM	NaCl	Sodium chloride

Details: 15 mM Hepes pH 7.4, 150 mM NaCl

• Grid: Model: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa / Details: Current: 30 mA
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287 K / Instrument: LEICA EM GP

Electron microscopy

• Microscope: TFS KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 22046 / Average electron dose: 46.6 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 2151332
• CTF correction: Software - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: OTHER / Details: Ab-initio model
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 7078
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
• Final 3D classification: Software - Name: RELION (ver. 3.1)

Atomic model buiding 1

Initial model

PDB ID:

6huo

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

• Refinement: Space: REAL / Protocol: OTHER / Overall B value: 30 / Target criteria: FSC at 0.5

Output model

PDB-9fam:
CryoEM structure of human full-length alpha1beta3gamma2 GABA(A)R in complex with GARLH4, the TMD of Neuroligin2, GABA and Megabody38 in a desensitised state (StateD3)