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- EMDB-44754: Cryo-EM structure of human Glycine Receptor apha3-beta heteromer ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44754
TitleCryo-EM structure of human Glycine Receptor apha3-beta heteromer with glycine in nanodisc
Map dataCryo-EM structure of human Glycine Receptor apha3-beta heteromer with glycine in nanodisc
Sample
  • Complex: heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc
    • Protein or peptide: Glycine receptor subunit alpha-3
    • Protein or peptide: Glycine receptor subunit beta,Green fluorescent protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: GLYCINE
Keywordsglycine receptor subunit alpha-3 / glycine receptor subunit beta / Green fluorescent protein / MEMBRANE PROTEIN
Function / homology
Function and homology information


synaptic transmission, glycinergic / glycine-gated chloride ion channel activity / acrosome reaction / glycine-gated chloride channel complex / gamma-aminobutyric acid receptor clustering / Neurotransmitter receptors and postsynaptic signal transmission / postsynaptic specialization / extracellularly glycine-gated ion channel activity / righting reflex / extracellularly glycine-gated chloride channel activity ...synaptic transmission, glycinergic / glycine-gated chloride ion channel activity / acrosome reaction / glycine-gated chloride channel complex / gamma-aminobutyric acid receptor clustering / Neurotransmitter receptors and postsynaptic signal transmission / postsynaptic specialization / extracellularly glycine-gated ion channel activity / righting reflex / extracellularly glycine-gated chloride channel activity / glycinergic synapse / adult walking behavior / glycine binding / startle response / neuropeptide signaling pathway / monoatomic ion transport / visual perception / chloride transmembrane transport / bioluminescence / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / GABA-ergic synapse / generation of precursor metabolites and energy / protein homooligomerization / transmembrane signaling receptor activity / nervous system development / chemical synaptic transmission / perikaryon / postsynaptic membrane / intracellular membrane-bounded organelle / dendrite / protein-containing complex binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
: / Glycine receptor alpha3 / Glycine receptor beta / : / Glycine receptor alpha / 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. ...: / Glycine receptor alpha3 / Glycine receptor beta / : / Glycine receptor alpha / 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 / Green fluorescent protein, GFP / 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 / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Green fluorescent protein / Glycine receptor subunit alpha-3 / Glycine receptor subunit beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.81 Å
AuthorsLiu X / Wang W
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)1R35GM146860 United States
CitationJournal: Nat Commun / Year: 2025
Title: Mechanism of human α3β GlyR regulation by intracellular M3/M4 loop phosphorylation and 2,6-di-tert-butylphenol interaction.
Authors: Xiaofen Liu / Malgorzata Krezel / Weiwei Wang /
Abstract: α3β glycine receptor (GlyR) is a subtype of GlyRs that belongs to the Cys-loop receptor superfamily. It is highly expressed in the spinal dorsal horn where sensory information is integrated. Under ...α3β glycine receptor (GlyR) is a subtype of GlyRs that belongs to the Cys-loop receptor superfamily. It is highly expressed in the spinal dorsal horn where sensory information is integrated. Under inflammatory conditions, the large unstructured intracellular M3/M4 loops of the α3 subunit are phosphorylated through the prostaglandin E2 (PGE) pathway, inhibiting ion conduction, and resulting in elevated pain sensation. A small molecule analgesic analog, 2,6-di-tert-butylphenol (2,6-DTBP) potentiates phosphorylated α3β GlyR through unclear mechanisms and relieves pain. Combining cryo-Electron Microscopy (cryo-EM) structures and single molecule Förster resonance energy transfer (smFRET) experiments, we show compaction of M3/M4 loop towards the ion conduction pore upon phosphorylation and further by 2,6-DTBP binding, which in turn modulates function through changing pore conformations and local electrostatics. We show that simultaneous interactions with the M3/M4 loop and the transmembrane domain (TM) is necessary for the potentiation of heteromeric α3β GlyR by 2,6-DTBP, while TM interaction alone is sufficient to potentiate homomeric α3 GlyR, explaining the mystery of why 2,6-DTBP potentiates only phosphorylated α3β GlyR. These findings show how post-translational modification of the unstructured intracellular M3/M4 loop may regulate Cys-loop receptor function, providing new perspectives in pain control and other pharmaceutical development targeting GlyRs and other Cys-loop receptors.
History
DepositionMay 6, 2024-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateJun 25, 2025-
Current statusJun 25, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44754.png

Downloads & links

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Map

FileDownload / File: emd_44754.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of human Glycine Receptor apha3-beta heteromer with glycine in nanodisc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.29
Minimum - Maximum-1.4793682 - 2.1308725
Average (Standard dev.)0.011954549 (±0.06895383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_44754_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_44754_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc

EntireName: heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc
Components
  • Complex: heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc
    • Protein or peptide: Glycine receptor subunit alpha-3
    • Protein or peptide: Glycine receptor subunit beta,Green fluorescent protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: GLYCINE

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Supramolecule #1: heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc

SupramoleculeName: heteromeric glycine receptor alpha-3 and beta with glycine in nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: Glycine receptor subunit alpha-3

MacromoleculeName: Glycine receptor subunit alpha-3 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.93652 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ARSRSAPMSP SDFLDKLMGR TSGYDARIRP NFKGPPVNVT CNIFINSFGS IAETTMDYRV NIFLRQKWND PRLAYSEYPD DSLDLDPSM LDSIWKPDLF FANEKGANFH EVTTDNKLLR IFKNGNVLYS IRLTLTLSCP MDLKNFPMDV QTCIMQLESF G YTMNDLIF ...String:
ARSRSAPMSP SDFLDKLMGR TSGYDARIRP NFKGPPVNVT CNIFINSFGS IAETTMDYRV NIFLRQKWND PRLAYSEYPD DSLDLDPSM LDSIWKPDLF FANEKGANFH EVTTDNKLLR IFKNGNVLYS IRLTLTLSCP MDLKNFPMDV QTCIMQLESF G YTMNDLIF EWQDEAPVQV AEGLTLPQFL LKEEKDLRYC TKHYNTGKFT CIEVRFHLER QMGYYLIQMY IPSLLIVILS WV SFWINMD AAPARVALGI TTVLTMTTQS SGSRASLPKV SYVKAIDIWM AVCLLFVFSA LLEYAAVNFV SRQHKELLRF RRK RKNKTE AFDMDDEVRE SRFSFTAYGM GPCLQAKDGM TPKGPNHPVQ VMPKSPDEMR KVFIDRAKKI DTISRACFPL AFLI FNIFY WVIYKILRHE DIHQQQD

UniProtKB: Glycine receptor subunit alpha-3

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Macromolecule #2: Glycine receptor subunit beta,Green fluorescent protein

MacromoleculeName: Glycine receptor subunit beta,Green fluorescent protein
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 76.584531 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KSSKKGKGKK KQYLCPSQQS AEDLARVPAN STSNILNRLL VSYDPRIRPN FKGIPVDVVV NIFINSFGSI QETTMDYRVN IFLRQKWND PRLKLPSDFR GSDALTVDPT MYKCLWKPDL FFANEKSANF HDVTQENILL FIFRDGDVLV SMRLSITLSC P LDLTLFPM ...String:
KSSKKGKGKK KQYLCPSQQS AEDLARVPAN STSNILNRLL VSYDPRIRPN FKGIPVDVVV NIFINSFGSI QETTMDYRVN IFLRQKWND PRLKLPSDFR GSDALTVDPT MYKCLWKPDL FFANEKSANF HDVTQENILL FIFRDGDVLV SMRLSITLSC P LDLTLFPM DTQRCKMQLE SFGYTTDDLR FIWQSGDPVQ LEKIALPQFD IKKEDIEYGN CTKYYKGTGY YTCVEVIFTL RR QVGFYMM GVYAPTLLIV VLSWLSFWIN PDASAARVPL GIFSVLSLAS ECTTLAAELP KVSYVKALDV WLIACLLFGF ASL VEYAVV QVMLNGGSSA AAVSKGEELF TGVVPILVEL DGDVNGHKFS VSGEGEGDAT YGKLTLKFIC TTGKLPVPWP TLVT TFSYG VQCFSRYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLE YNYN SHNVYIMADK QKNGIKVNFK IRHNIEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSA LSKDPNEKRD HMVLLE FVT AAGITHGMDE LYKSGSGSGV GETRCKKVCT SKSDLRSNDF SIVGSLPRDF ELSNYDCYGK PIEVNNGLGK SQAKNNK KP PPAKPVIPTA AKRIDLYARA LFPFCFLFFN VIYWSIYL

UniProtKB: Glycine receptor subunit beta, Green fluorescent protein, Glycine receptor subunit beta

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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: 5 / 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: GLYCINE

MacromoleculeName: GLYCINE / type: ligand / ID: 4 / Number of copies: 5 / Formula: GLY
Molecular weightTheoretical: 75.067 Da
Chemical component information

ChemComp-GLY:
GLYCINE

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
20.0 mMTris-HClTris-HCl
GridModel: Quantifoil R1.2/1.3 / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 69.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8dn4

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.81 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 40868
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

8dn4


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

PDB-9boy:
Cryo-EM structure of human Glycine Receptor apha3-beta heteromer with glycine in nanodisc

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