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- EMDB-27968: CryoEM structure of miniGq-coupled hM3Dq in complex with CNO -

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Basic information

Entry
Database: EMDB / ID: EMD-27968
TitleCryoEM structure of miniGq-coupled hM3Dq in complex with CNO
Map dataCryoEM structure of Gq-coupled hM3RD in complex with CNO
Sample
  • Complex: Gq-coupled hM3R complex
    • Complex: muscarinic acetylcholine receptor 3, miniGq protein, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Muscarinic acetylcholine receptor M3
      • Protein or peptide: miniGq
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Complex: Single-chain variable fragment scFv16
      • Protein or peptide: scFv16
  • Ligand: 8-chloro-11-(4-methyl-4-oxo-4lambda~5~-piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine
Function / homology
Function and homology information


regulation of monoatomic ion transmembrane transporter activity / regulation of vascular associated smooth muscle contraction / saliva secretion / Acetylcholine regulates insulin secretion / Muscarinic acetylcholine receptors / ion channel modulating, G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / positive regulation of smooth muscle contraction ...regulation of monoatomic ion transmembrane transporter activity / regulation of vascular associated smooth muscle contraction / saliva secretion / Acetylcholine regulates insulin secretion / Muscarinic acetylcholine receptors / ion channel modulating, G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / positive regulation of smooth muscle contraction / phosphatidylinositol phospholipase C activity / G protein-coupled serotonin receptor activity / acetylcholine binding / acetylcholine receptor signaling pathway / ligand-gated ion channel signaling pathway / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / smooth muscle contraction / basal plasma membrane / calcium-mediated signaling / Olfactory Signaling Pathway / Activation of the phototransduction cascade / protein modification process / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / positive regulation of insulin secretion / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / signaling receptor activity / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / nervous system development / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / chemical synaptic transmission / G alpha (q) signalling events / postsynaptic membrane / basolateral plasma membrane / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / dendrite / synapse / protein-containing complex binding / endoplasmic reticulum membrane / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Muscarinic acetylcholine receptor M3 / Muscarinic acetylcholine receptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs ...Muscarinic acetylcholine receptor M3 / Muscarinic acetylcholine receptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Muscarinic acetylcholine receptor M3 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsZhang S / Fay JF / Roth BL
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)RO1MH112205 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)U24DK1169195 United States
CitationJournal: Nature / Year: 2022
Title: Molecular basis for selective activation of DREADD-based chemogenetics.
Authors: Shicheng Zhang / Ryan H Gumpper / Xi-Ping Huang / Yongfeng Liu / Brian E Krumm / Can Cao / Jonathan F Fay / Bryan L Roth /
Abstract: Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling. The muscarinic ...Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling. The muscarinic receptor-based DREADDs are the most widely used chemogenetic tools in neuroscience research. The G-coupled DREADD (hM3Dq) is used to enhance neuronal activity, whereas the G-coupled DREADD (hM4Di) is utilized to inhibit neuronal activity. Here we report four DREADD-related cryogenic electron microscopy high-resolution structures: a hM3Dq-miniG complex and a hM4Di-miniG complex bound to deschloroclozapine; a hM3Dq-miniG complex bound to clozapine-N-oxide; and a hM3R-miniG complex bound to iperoxo. Complemented with mutagenesis, functional and computational simulation data, our structures reveal key details of the recognition of DREADD chemogenetic actuators and the molecular basis for activation. These findings should accelerate the structure-guided discovery of next-generation chemogenetic tools.
History
DepositionAug 27, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateDec 21, 2022-
Current statusDec 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27968.png

Downloads & links

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Map

FileDownload / File: emd_27968.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of Gq-coupled hM3RD in complex with CNO
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.022526884 - 2.3228626
Average (Standard dev.)0.001101947 (±0.021207083)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 253.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: raw

Fileemd_27968_additional_1.map
Annotationraw
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_27968_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_27968_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Gq-coupled hM3R complex

EntireName: Gq-coupled hM3R complex
Components
  • Complex: Gq-coupled hM3R complex
    • Complex: muscarinic acetylcholine receptor 3, miniGq protein, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Muscarinic acetylcholine receptor M3
      • Protein or peptide: miniGq
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Complex: Single-chain variable fragment scFv16
      • Protein or peptide: scFv16
  • Ligand: 8-chloro-11-(4-methyl-4-oxo-4lambda~5~-piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine

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Supramolecule #1: Gq-coupled hM3R complex

SupramoleculeName: Gq-coupled hM3R complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: muscarinic acetylcholine receptor 3, miniGq protein, Guanine nucl...

SupramoleculeName: muscarinic acetylcholine receptor 3, miniGq protein, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Single-chain variable fragment scFv16

SupramoleculeName: Single-chain variable fragment scFv16 / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Muscarinic acetylcholine receptor M3

MacromoleculeName: Muscarinic acetylcholine receptor M3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 62.892555 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AGNFSSPDGT TDDPLGGHTV WQVVFIAFLT GILALVTIIG NILVIVSFKV NKQLKTVNNY FLLSLACADL IIGVISMNLF TTYIIMNRW ALGNLACDLW LAIDCVASNA SVMNLLVISF DRYFSITRPL TYRAKRTTKR AGVMIGLAWV ISFVLWAPAI L FWQYFVGK ...String:
AGNFSSPDGT TDDPLGGHTV WQVVFIAFLT GILALVTIIG NILVIVSFKV NKQLKTVNNY FLLSLACADL IIGVISMNLF TTYIIMNRW ALGNLACDLW LAIDCVASNA SVMNLLVISF DRYFSITRPL TYRAKRTTKR AGVMIGLAWV ISFVLWAPAI L FWQYFVGK RTVPPGECFI QFLSEPTITF GTAIAGFYMP VTIMTILYWR IYKETEKRTK ELAGLQASGT EAETENFVHP AK RFALKTR SQITKRKRMS LVKEKKAAQT LSAILLAFII TWTPYNIMVL VNTFCDSCIP KTFWNLGYWL CYINSTVNPV CYA LCNKTF RTTFKMLLLC QCDKKKRRKQ QYQQRQSVIF HKRAPEQALG GSGGGGSGGS SSGGGGSGGG GSGGSSSGGV FTLE DFVGD WEQTAAYNLD QVLEQGGVSS LLQNLAVSVT PIQRIVRSGE NALKIDIHVI IPYEGLSADQ MAQIEEVFKV VYPVD DHHF KVILPYGTLV IDGVTPNMLN YFGRPYEGIA VFDGKKITVT GTLWNGNKII DERLITPDGS MLFRVTINSG GSGGHH HHH HHHHH

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Macromolecule #2: miniGq

MacromoleculeName: miniGq / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.084832 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSTVSAEDK AAAERSKMID KNLREDGEKA RRTLRLLLLG ADNSGKSTIV KQMRILHGGS GGSGGTSGIF ETKFQVDKVN FHMFDVGGQ RDERRKWIQC FNDVTAIIFV VDSSDYNRLQ EALNDFKSIW NNRWLRTISV ILFLNKQDLL AEKVLAGKSK I EDYFPEFA ...String:
MGSTVSAEDK AAAERSKMID KNLREDGEKA RRTLRLLLLG ADNSGKSTIV KQMRILHGGS GGSGGTSGIF ETKFQVDKVN FHMFDVGGQ RDERRKWIQC FNDVTAIIFV VDSSDYNRLQ EALNDFKSIW NNRWLRTISV ILFLNKQDLL AEKVLAGKSK I EDYFPEFA RYTTPEDATP EPGEDPRVTR AKYFIRKEFV DISTASGDGR HICYPHFTCA VDTENARRIF NDCKDIILQM NL REYNLV

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.79841 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLII WDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR FLDDNQIVTS S GDTTCALW ...String:
SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLII WDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR FLDDNQIVTS S GDTTCALW DIETGQQTTT FTGHTGDVMS LSLAPDTRLF VSGACDASAK LWDVREGMCR QTFTGHESDI NAICFFPNGN AF ATGSDDA TCRLFDLRAD QELMTYSHDN IICGITSVSF SKSGRLLLAG YDDFNCNVWD ALKADRAGVL AGHDNRVSCL GVT DDGMAV ATGSWDSFLK IWNGGSGGGG SGGSSSGGVS GWRLFKKISG GS

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Macromolecule #4: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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Macromolecule #5: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.679721 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS ...String:
DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LEAEDVGVYY CMQHLEYPLT FG AGTKLEL KAAA

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Macromolecule #6: 8-chloro-11-(4-methyl-4-oxo-4lambda~5~-piperazin-1-yl)-5H-dibenzo...

MacromoleculeName: 8-chloro-11-(4-methyl-4-oxo-4lambda~5~-piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine
type: ligand / ID: 6 / Number of copies: 1 / Formula: WE9
Molecular weightTheoretical: 342.823 Da
Chemical component information

ChemComp-WE9:
8-chloro-11-(4-methyl-4-oxo-4lambda~5~-piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine / Clozapine N-oxide

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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-PROPANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.4 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3577 / Average electron dose: 46.96 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6oij

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 579660
FSC plot (resolution estimation)

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