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- EMDB-52411: Inward-open structure of human glycine transporter 2 in substrate... -

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Basic information

Entry
Database: EMDB / ID: EMD-52411
TitleInward-open structure of human glycine transporter 2 in substrate-free state
Map dataSubstrate-free GlyT2, Final map
Sample
  • Complex: Human glycine transporter GlyT2 in substrate-free state
    • Protein or peptide: Sodium- and chloride-dependent glycine transporter 2
  • Ligand: CHOLESTEROL
  • Ligand: CHLORIDE ION
KeywordsTransport protein / SLC6A5 / neurotransmitter/sodium symporter / Sodium- and chloride-dependent glycine transporter 2 / MEMBRANE PROTEIN
Function / homology
Function and homology information


Defective SLC6A5 causes hyperekplexia 3 (HKPX3) / glycine:sodium symporter activity / synaptic transmission, glycinergic / glycine import across plasma membrane / dense core granule / SLC-mediated transport of neurotransmitters / neurotransmitter transport / sodium ion transmembrane transport / chemical synaptic transmission / endosome ...Defective SLC6A5 causes hyperekplexia 3 (HKPX3) / glycine:sodium symporter activity / synaptic transmission, glycinergic / glycine import across plasma membrane / dense core granule / SLC-mediated transport of neurotransmitters / neurotransmitter transport / sodium ion transmembrane transport / chemical synaptic transmission / endosome / synapse / membrane / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
Sodium- and chloride-dependent glycine transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsCantwell Chater RP / Peiser-Oliver J / Pati TK / Quinn AS / Lotsaris I / Frangos ZJ / Anderson KE / Tischer AE / Williams-Noonan BJ / Aubrey KR ...Cantwell Chater RP / Peiser-Oliver J / Pati TK / Quinn AS / Lotsaris I / Frangos ZJ / Anderson KE / Tischer AE / Williams-Noonan BJ / Aubrey KR / O Mara ML / Michaelides M / Mohammadi SA / Cioffi CL / Vandenberg RJ / Shahsavar A
Funding support Denmark, United States, 3 items
OrganizationGrant numberCountry
LundbeckfondenR368-2021-522 Denmark
Novo Nordisk FoundationNNF23OC0087107 Denmark
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)R01DA048879 United States
CitationJournal: Nat Commun / Year: 2026
Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects.
Authors: Ryan P Cantwell Chater / Julian Peiser-Oliver / Tanmay K Pati / Ada S Quinn / Irina Lotsaris / Zachary J Frangos / Kristen E Anderson / Anna E Tischer / Billy J Williams-Noonan / Karin R ...Authors: Ryan P Cantwell Chater / Julian Peiser-Oliver / Tanmay K Pati / Ada S Quinn / Irina Lotsaris / Zachary J Frangos / Kristen E Anderson / Anna E Tischer / Billy J Williams-Noonan / Karin R Aubrey / Megan L O'Mara / Michael Michaelides / Sarasa A Mohammadi / Christopher L Cioffi / Robert J Vandenberg / Azadeh Shahsavar /
Abstract: Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, ...Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2-82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2-82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2-82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.
History
DepositionDec 22, 2024-
Header (metadata) releaseFeb 25, 2026-
Map releaseFeb 25, 2026-
UpdateApr 8, 2026-
Current statusApr 8, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52411.png

Downloads & links

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Map

FileDownload / File: emd_52411.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubstrate-free GlyT2, Final map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 300 pix.
= 218.4 Å		0.73 Å/pix.
x 300 pix.
= 218.4 Å		0.73 Å/pix.
x 300 pix.
= 218.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.728 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.034
Minimum - Maximum-0.24286063 - 0.36734807
Average (Standard dev.)0.00025502092 (±0.008360533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 218.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_52411_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_52411_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 : Human glycine transporter GlyT2 in substrate-free state

EntireName: Human glycine transporter GlyT2 in substrate-free state
Components
  • Complex: Human glycine transporter GlyT2 in substrate-free state
    • Protein or peptide: Sodium- and chloride-dependent glycine transporter 2
  • Ligand: CHOLESTEROL
  • Ligand: CHLORIDE ION

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Supramolecule #1: Human glycine transporter GlyT2 in substrate-free state

SupramoleculeName: Human glycine transporter GlyT2 in substrate-free state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Sodium- and chloride-dependent glycine transporter 2

MacromoleculeName: Sodium- and chloride-dependent glycine transporter 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.976023 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDENKARGNW SSKLDFILSM VGYAVGLGNV WRFPYLAFQN GGGAFLIPYL MMLALAGLPI FFLEVSLGQF ASQGPVSVWK AIPALQGCG IAMLIISVLI AIYYNVIICY TLFYLFASFV SVLPWGSCNN PWNTPECKDK TKLLLDSCVI SDHPKIQIKN S TFCMTAYP ...String:
MDENKARGNW SSKLDFILSM VGYAVGLGNV WRFPYLAFQN GGGAFLIPYL MMLALAGLPI FFLEVSLGQF ASQGPVSVWK AIPALQGCG IAMLIISVLI AIYYNVIICY TLFYLFASFV SVLPWGSCNN PWNTPECKDK TKLLLDSCVI SDHPKIQIKN S TFCMTAYP NVTMVNFTSQ ANKTFVSGSE EYFKYFVLKI SAGIEYPGEI RWPLALCLFL AWVIVYASLA KGIKTSGKVV YF TATFPYV VLVILLIRGV TLPGAGAGIW YFITPKWEKL TDATVWKDAA TQIFFSLSAA WGGLITLSSY NKFHNNCYRD TLI VTCTNS ATSIFAGFVI FSVIGFMANE RKVNIENVAD QGPGIAFVVY PEALTRLPLS PFWAIIFFLM LLTLGLDTMF ATIE TIVTS ISDEFPKYLR THKPVFTLGC CICFFIMGFP MITQGGIYMF QLVDTYAASY ALVIIAIFEL VGISYVYGLQ RFCED IEMM IGFQPNIFWK VCWAFVTPTI LTFILCFSFY QWEPMTYGSY RYPNWSMVLG WLMLACSVIW IPIMFVIKMH LAPGRF IER LKLVCSPQPD WGPFLAQHRG ERYKNMIDPL GTSSLGLKLP VKDLELGTQC

UniProtKB: Sodium- and chloride-dependent glycine transporter 2

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 21335 / Average exposure time: 3.34 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 165000
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: 6458661
CTF correctionType: NONE
Startup modelType of model: OTHER / Details: AF-Q9Y345-F1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 180698
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5-f1


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: UCSF ChimeraX (ver. 1.8)
Output model

PDB-9hug:
Inward-open structure of human glycine transporter 2 in substrate-free state

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