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- EMDB-48828: Focused refinement map of hM4Di(GRANPA) in complex with mGsI and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-48828
TitleFocused refinement map of hM4Di(GRANPA) in complex with mGsI and diphenhydramine
Map data
Sample
  • Complex: Human M4Di(GRANPA)-miniGsI-Gb1g2 complex
KeywordsG protein-coupled receptor / acetylcholine binding / seven transmembrane protein / MEMBRANE PROTEIN / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMobbs JI / Thal DM
Funding support Australia, 1 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1196951 Australia
CitationJournal: Signal Transduct Target Ther / Year: 2026
Title: Next-generation chemogenetic inhibition using a brain-permeant non-prescription agent.
Authors: Steven O Devenish / Sahil D Patel / Laura V Ussingkær / Luiz F Almeida Silva / Olivia Goff / Amy Richardson / Jesse I Mobbs / Hariprasad Venugopal / David M Thal / Dimitri M Kullmann /
Abstract: Chemogenetics allows the controllable manipulation of brain circuits upon delivery of a selective activating ligand, and has been invaluable in dissecting brain circuits underlying many behaviours. ...Chemogenetics allows the controllable manipulation of brain circuits upon delivery of a selective activating ligand, and has been invaluable in dissecting brain circuits underlying many behaviours. The Gαi/o-coupled designer muscarinic receptor hM4Di is an especially versatile tool for on-demand inhibition, and has proven effective not only in fundamental neuroscience but also as a therapeutic transgene in preclinical models of epilepsy and other CNS disorders. Indeed, by placing the circuit modulation under the control of an exogenous ligand, chemogenetics mitigates the potential risk of overdosage intrinsic to viral-vector mediated gene therapy. An obstacle to clinical translation, however, is the absence of an activating ligand with favourable biodistribution and side effect profile. Here we show that mutation of hM4Di at two sites (S85 and Y416) imparts full and potent agonism to the widely used over-the-counter antihistamine diphenhydramine. We complement medium-throughput screening in human embryonic kidney cells with in vitro electrophysiological characterization in neuronal circuits, and reveal the interaction of diphenhydramine with key residues using cryo-electron microscopy. Administration of diphenhydramine to mice expressing the modified receptor in the ventral hippocampus reversibly modulated anxiety-related behaviour and attenuated the severity of chemoconvulsant-induced seizures. We further demonstrate on-demand seizure suppression in a chronic epilepsy model. G protein-coupled Receptors Activated by Non-Prescription Agents (GRANPAs) lower the barrier to clinical translation of a powerful chemogenetic approach to brain circuit manipulation.
History
DepositionJan 28, 2025-
Header (metadata) releaseJul 15, 2026-
Map releaseJul 15, 2026-
UpdateJul 15, 2026-
Current statusJul 15, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48828.png

Downloads & links

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Map

FileDownload / File: emd_48828.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.31
Minimum - Maximum-0.9664329 - 1.6548301
Average (Standard dev.)0.00042975674 (±0.0266183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48828_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Half map: #1

Fileemd_48828_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_48828_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Histogram (log scale)

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Sample components

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Entire : Human M4Di(GRANPA)-miniGsI-Gb1g2 complex

EntireName: Human M4Di(GRANPA)-miniGsI-Gb1g2 complex
Components
  • Complex: Human M4Di(GRANPA)-miniGsI-Gb1g2 complex

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Supramolecule #1: Human M4Di(GRANPA)-miniGsI-Gb1g2 complex

SupramoleculeName: Human M4Di(GRANPA)-miniGsI-Gb1g2 complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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 / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
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: INSILICO MODEL / In silico model: ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 189628
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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