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- EMDB-0993: Cryo-EM map of PAC1 receptor bound to PACAP38 and Gs protein -

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Basic information

Entry
Database: EMDB / ID: EMD-0993
TitleCryo-EM map of PAC1 receptor bound to PACAP38 and Gs protein
Map dataSharpened map of PAC1 receptor bound to PACAP38 and Gs protein
Sample
  • Complex: PAC1 receptor bound to PACAP38 and Gs protein
Function / homology
Function and homology information


negative regulation of response to reactive oxygen species / development of primary female sexual characteristics / vasoactive intestinal polypeptide receptor activity / NGF-independant TRKA activation / neuropeptide binding / G protein-coupled peptide receptor activity / positive regulation of small GTPase mediated signal transduction / positive regulation of inositol phosphate biosynthetic process / positive regulation of calcium ion transport into cytosol / PKA activation in glucagon signalling ...negative regulation of response to reactive oxygen species / development of primary female sexual characteristics / vasoactive intestinal polypeptide receptor activity / NGF-independant TRKA activation / neuropeptide binding / G protein-coupled peptide receptor activity / positive regulation of small GTPase mediated signal transduction / positive regulation of inositol phosphate biosynthetic process / positive regulation of calcium ion transport into cytosol / PKA activation in glucagon signalling / peptide hormone binding / cAMP-mediated signaling / hair follicle placode formation / adenylate cyclase binding / intracellular transport / D1 dopamine receptor binding / developmental growth / bicellular tight junction / multicellular organismal response to stress / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / caveola / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / small GTPase binding / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / 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 / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / response to estradiol / retina development in camera-type eye / signaling receptor activity / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / spermatogenesis / response to ethanol / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / cell differentiation / cell surface receptor signaling pathway / receptor complex / endosome / response to xenobiotic stimulus / G protein-coupled receptor signaling pathway / lysosomal membrane / intracellular membrane-bounded organelle / GTPase activity / synapse / protein-containing complex binding
Similarity search - Function
GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like ...GPCR, family 2, pituitary adenylate cyclase activating polypeptide type 1 receptor / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / 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 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Pituitary adenylate cyclase-activating polypeptide type I receptor / 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 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsLiang YL / Belousoff MJ / Zhao P / Danev R / Sexton PM / Wootten D
Funding support Australia, Japan, 5 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1120919 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
National Health and Medical Research Council (NHMRC, Australia)1159006 Australia
Japan Science and Technology18069571 Japan
Japan Society for the Promotion of Science (JSPS)18H06043 Japan
CitationJournal: Mol Cell / Year: 2020
Title: Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation.
Authors: Yi-Lynn Liang / Matthew J Belousoff / Peishen Zhao / Cassandra Koole / Madeleine M Fletcher / Tin T Truong / Villy Julita / George Christopoulos / H Eric Xu / Yan Zhang / Maryam Khoshouei / ...Authors: Yi-Lynn Liang / Matthew J Belousoff / Peishen Zhao / Cassandra Koole / Madeleine M Fletcher / Tin T Truong / Villy Julita / George Christopoulos / H Eric Xu / Yan Zhang / Maryam Khoshouei / Arthur Christopoulos / Radostin Danev / Patrick M Sexton / Denise Wootten /
Abstract: Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, ...Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.
History
DepositionFeb 3, 2020-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0993.png

Downloads & links

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Map

FileDownload / File: emd_0993.map.gz / Format: CCP4 / Size: 20.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of PAC1 receptor bound to PACAP38 and Gs protein
Voxel sizeX=Y=Z: 1.207 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.20266752 - 0.37457576
Average (Standard dev.)0.00000797852 (±0.011056929)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 212.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2071.2071.207
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z212.432212.432212.432
α/β/γ90.00090.00090.000
start NX/NY/NZ-31-35-52
NX/NY/NZ11798209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.2030.3750.000

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Supplemental data

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Mask #1

Fileemd_0993_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unprocessed map of PAC1 receptor bound to PACAP38 and Gs protein

Fileemd_0993_additional.map
AnnotationUnprocessed map of PAC1 receptor bound to PACAP38 and Gs protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unprocessed map of PAC1 receptor bound to PACAP38 and Gs protein

Fileemd_0993_additional_1.map
AnnotationUnprocessed map of PAC1 receptor bound to PACAP38 and Gs protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unprocessed half-map of PAC1 receptor bound to PACAP38 and Gs protein

Fileemd_0993_half_map_1.map
AnnotationUnprocessed half-map of PAC1 receptor bound to PACAP38 and Gs protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unprocessed half-map of PAC1 receptor bound to PACAP38 and Gs protein

Fileemd_0993_half_map_2.map
AnnotationUnprocessed half-map of PAC1 receptor bound to PACAP38 and Gs protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PAC1 receptor bound to PACAP38 and Gs protein

EntireName: PAC1 receptor bound to PACAP38 and Gs protein
Components
  • Complex: PAC1 receptor bound to PACAP38 and Gs protein

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Supramolecule #1: PAC1 receptor bound to PACAP38 and Gs protein

SupramoleculeName: PAC1 receptor bound to PACAP38 and Gs protein / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 150 kDa/nm

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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

Concentration4.35 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot time 10 s.

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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 / Calibrated defocus max: 1.6 µm / Calibrated defocus min: 0.4 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 105000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 25 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7650 / Average exposure time: 3.715 sec. / Average electron dose: 64.0 e/Å2
Details: Volta phase plate images: 4032; Conventional defocus images: 3618
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5500000
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: 20A low-pass filtered GPCR map from previous experiment.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Software - details: 3
Final 3D classificationNumber classes: 3 / Software - Name: RELION / Software - details: 3
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Software - details: 3
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Software - details: 3 / Number images used: 390000

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