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- EMDB-14067: Cryo-EM structure of human full-length extrasynaptic alpha4beta3d... -

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Basic information

Entry
Database: EMDB / ID: EMD-14067
TitleCryo-EM structure of human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with nanobody Nb25
Map data
Sample
  • Complex: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with Nanobody Nb25
    • Protein or peptide: x 4 types
  • Ligand: x 7 types
Function / homology
Function and homology information


benzodiazepine receptor activity / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / postsynaptic specialization membrane / neurotransmitter receptor activity ...benzodiazepine receptor activity / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / postsynaptic specialization membrane / neurotransmitter receptor activity / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / chloride channel activity / roof of mouth development / Signaling by ERBB4 / chloride channel complex / GABA-ergic synapse / regulation of postsynaptic membrane potential / chloride transmembrane transport / dendrite membrane / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / postsynapse / postsynaptic membrane / neuron projection / axon / neuronal cell body / dendrite / synapse / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region ...Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / 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
Similarity search - Domain/homology
Gamma-aminobutyric acid receptor subunit delta / Gamma-aminobutyric acid receptor subunit beta-3 / Gamma-aminobutyric acid receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsSente A / Desai R / Naydenova K / Malinauskas T / Jounaidi Y / Miehling J / Zhou X / Masiulis S / Hardwick SW / Chirgadze DY ...Sente A / Desai R / Naydenova K / Malinauskas T / Jounaidi Y / Miehling J / Zhou X / Masiulis S / Hardwick SW / Chirgadze DY / Miller KW / Aricescu AR
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01-GM135550 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z United Kingdom
CitationJournal: Nature / Year: 2022
Title: Differential assembly diversifies GABA receptor structures and signalling.
Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith ...Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith W Miller / A Radu Aricescu /
Abstract: Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines ...Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines including general anaesthetics and benzodiazepines. Human GABAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABARs assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABARs.
History
DepositionDec 20, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateMay 4, 2022-
Current statusMay 4, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14067.png

Downloads & links

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Map

FileDownload / File: emd_14067.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 290 pix.
= 259.84 Å		0.9 Å/pix.
x 290 pix.
= 259.84 Å		0.9 Å/pix.
x 290 pix.
= 259.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.896 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0085
Minimum - Maximum-0.03443107 - 0.09564092
Average (Standard dev.)-8.957971e-05 (±0.0026736313)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 259.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human full-length extrasynaptic alpha4beta3delta GABA(A)R in comp...

EntireName: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with Nanobody Nb25
Components
  • Complex: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with Nanobody Nb25
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-4
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit delta
    • Protein or peptide: Nanobody Nb25
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE
  • Ligand: DECANE
  • Ligand: N-OCTANEOctane
  • Ligand: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID
  • Ligand: CHLORIDE IONChloride
  • Ligand: water

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Supramolecule #1: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in comp...

SupramoleculeName: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with Nanobody Nb25
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S TetR

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit alpha-4

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.696047 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGPVSDV EMEYTMDVFF RQTWIDKRLK YDGPIEILRL NNMMVTKVWT PDTFFRNGKK SVSHNMTAPN KLFRIMRNGT I LYTMRLTI ...String:
MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGPVSDV EMEYTMDVFF RQTWIDKRLK YDGPIEILRL NNMMVTKVWT PDTFFRNGKK SVSHNMTAPN KLFRIMRNGT I LYTMRLTI SAECPMRLVD FPMDGHACPL KFGSYAYPKS EMIYTWTKGP EKSVEVPKES SSLVQYDLIG QTVSSETIKS IT GEYIVMT VYFHLRRKMG YFMIQTYIPC IMTVILSQVS FWINKESVPA RTVFGITTVL TMTTLSISAR HSLPKVSYAT AMD WFIAVC FAFVFSALIE FAAVNYFTNI QMEKAKRKTS KPPQEVPAAP VQREKHPEAP LQNTNANLNM RKRTNALVHS ESDV GNRTE VGNHSSKSST VVQESSKGTP RSYLASSPNP FSRANAAETI SAARALPSAS PTSIRTGYMP RKASVGSAST RHVFG SRLQ RIKTTVNTIG ATGKLSATPP PSAPPPSGSG TSKIDKYARI LFPVTFGAFN MVYWVVYLSK DTMEKSESLM

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

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-3 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.180348 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWGLAGGRLF GIFSAPVLVA VVCCAQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGPP VCVGMNIDIA SIDMVSEVNM DYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY ...String:
MWGLAGGRLF GIFSAPVLVA VVCCAQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGPP VCVGMNIDIA SIDMVSEVNM DYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY PLDEQNCTLE IESYGYTTDD IEFYWRGGDK AVTGVERIEL PQFSIVEHRL VSRNVVFATG AYPRLSLSFR LK RNIGYFI LQTYMPSILI TILSWVSFWI NYDASAARVA LGITTVLTMT TINTHLRETL PKIPYVKAID MYLMGCFVFV FLA LLEYAF VNYIFFGRGP QRQKKLAEKT AKAKNDRSKS ESNRVDAHGN ILLTSLEVHN EMNEVSGGIG DTRNSAISFD NSGI QYRKQ SMPREGHGRF LGDRSLPHKK THLRRRSSQL KIKIPDLTDV NAIDRWSRIV FPFTFSLFNL VYWLYYVN

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Macromolecule #3: Gamma-aminobutyric acid receptor subunit delta

MacromoleculeName: Gamma-aminobutyric acid receptor subunit delta / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.437926 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAPARLLAP LLLLCAQQLR GTRAMNDIGD YVGSNLEISW LPNLDGLIAG YARNFRPGIG GPPVNVALAL EVASIDHISE ANMEYTMTV FLHQSWRDSR LSYNHTNETL GLDSRFVDKL WLPDTFIVNA KSAWFHDVTV ENKLIRLQPD GVILYSIRIT S TVACDMDL ...String:
MDAPARLLAP LLLLCAQQLR GTRAMNDIGD YVGSNLEISW LPNLDGLIAG YARNFRPGIG GPPVNVALAL EVASIDHISE ANMEYTMTV FLHQSWRDSR LSYNHTNETL GLDSRFVDKL WLPDTFIVNA KSAWFHDVTV ENKLIRLQPD GVILYSIRIT S TVACDMDL AKYPMDEQEC MLDLESYGYS SEDIVYYWSE SQEHIHGLDK LQLAQFTITS YRFTTELMNF KSAGQFPRLS LH FHLRRNR GVYIIQSYMP SVLLVAMSWV SFWISQAAVP ARVSLGITTV LTMTTLMVSA RSSLPRASAI KALDVYFWIC YVF VFAALV EYAFAHFNAD YRKKQKAKVK VSRPRAEMDV RNAIVLFSLS AAGVTQELAI SRRQRRVPGN LMGSYRSVGV ETGE TKKEG AARSGGQGGI RARLRPIDAD TIDIYARAVF PAAFAAVNVI YWAAYAMGGS GGSGGSGKTE TSQVAPA

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Macromolecule #4: Nanobody Nb25

MacromoleculeName: Nanobody Nb25 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.341741 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQGSLRLSC AASGHTFNYP IMGWFRQAPG KEREFVGAIS WSGGSTSYAD SVKDRFTISR DNAKNTVYLE MNNLKPEDT AVYYCAAKGR YSGGLYYPTN YDYWGQGTQV TV

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 3 / 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 #8: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: PX6
Molecular weightTheoretical: 647.883 Da
Chemical component information

ChemComp-PX6:
1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

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Macromolecule #9: DECANE

MacromoleculeName: DECANE / type: ligand / ID: 9 / Number of copies: 2 / Formula: D10
Molecular weightTheoretical: 142.282 Da
Chemical component information

ChemComp-D10:
DECANE / Decane

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Macromolecule #10: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 10 / Number of copies: 2 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE / Octane

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Macromolecule #11: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID

MacromoleculeName: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / type: ligand / ID: 11 / Number of copies: 2 / Formula: EPE
Molecular weightTheoretical: 238.305 Da
Chemical component information

ChemComp-EPE:
4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / pH buffer*YM / HEPES

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Macromolecule #12: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 12 / Number of copies: 3 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #13: water

MacromoleculeName: water / type: ligand / ID: 13 / Number of copies: 174 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 7.6
Component:
ConcentrationFormulaName
300.0 mMNaClSodium chlorideSodium chloride
50.0 mMHEPES
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 30 mA (EasyGlow)
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Startup modelType of model: OTHER / Details: AB INITIO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 68547

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7qn5:
Cryo-EM structure of human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with nanobody Nb25

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