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- EMDB-13454: Active Melanocortin-4 receptor (MC4R)- Gs protein complex bound t... -

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Entry
Database: EMDB / ID: EMD-13454
TitleActive Melanocortin-4 receptor (MC4R)- Gs protein complex bound to agonist NDP-alpha-MSH at 2.86 A resolution.
Map data
Sample
  • Complex: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 receptor (MC4R) Gs protein complex stabilized by nanobody 35
    • Protein or peptide: Melanocortin receptor 4
    • Protein or peptide: NDP-alpha-MSH (other names Afamelanotide; Scenesse)
    • Protein or peptide: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • 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
    • Protein or peptide: Camelid antibody VHH fragment - nanobody 35
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
Function / homology
Function and homology information


regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma ...regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thromboxane signalling through TP receptor / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Ca2+ pathway / G alpha (s) signalling events / G alpha (q) signalling events / Extra-nuclear estrogen signaling / G alpha (12/13) signalling events / regulation of grooming behavior / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (i) signalling events / GPER1 signaling / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADP signalling through P2Y purinoceptor 1 / energy reserve metabolic process / neuropeptide binding / regulation of metabolic process / G-protein activation / Activation of the phototransduction cascade / : / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Activation of G protein gated Potassium channels / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (z) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / feeding behavior / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / alkylglycerophosphoethanolamine phosphodiesterase activity / insulin secretion / photoreceptor outer segment membrane / spectrin binding / diet induced thermogenesis / negative regulation of feeding behavior / PKA activation in glucagon signalling / peptide hormone binding / hair follicle placode formation / intracellular transport / photoreceptor outer segment / D1 dopamine receptor binding / developmental growth / positive regulation of bone resorption / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / cardiac muscle cell apoptotic process / activation of adenylate cyclase activity / photoreceptor inner segment / adenylate cyclase activator activity / Peptide ligand-binding receptors / trans-Golgi network membrane / G-protein beta/gamma-subunit complex binding / response to insulin / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / platelet aggregation / 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 / cellular response to prostaglandin E stimulus / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding
Similarity search - Function
Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 ...Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Melanocortin receptor 4 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others) / Rattus norvegicus (Norway rat) / Mus musculus (house mouse) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsHeyder NA / Schmidt A / Kleinau G / Hilal T / Scheerer P
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)CRC1423, project number 421152132, subprojects A01, A05, Z03, B02, C01, Z04 Germany
German Research Foundation (DFG)CRC1365, project number 394046635, subproject A03 Germany
German Research Foundation (DFG)Excellence Strategies - EXC2008/1 (UniSysCat) - 390540038 Germany
CitationJournal: Cell Res / Year: 2021
Title: Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-α-MSH and setmelanotide.
Authors: Nicolas A Heyder / Gunnar Kleinau / David Speck / Andrea Schmidt / Sarah Paisdzior / Michal Szczepek / Brian Bauer / Anja Koch / Monique Gallandi / Dennis Kwiatkowski / Jörg Bürger / ...Authors: Nicolas A Heyder / Gunnar Kleinau / David Speck / Andrea Schmidt / Sarah Paisdzior / Michal Szczepek / Brian Bauer / Anja Koch / Monique Gallandi / Dennis Kwiatkowski / Jörg Bürger / Thorsten Mielke / Annette G Beck-Sickinger / Peter W Hildebrand / Christian M T Spahn / Daniel Hilger / Magdalena Schacherl / Heike Biebermann / Tarek Hilal / Peter Kühnen / Brian K Kobilka / Patrick Scheerer /
Abstract: The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of ...The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.
History
DepositionAug 23, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-7piv
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13454.png

Downloads & links

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Map

FileDownload / File: emd_13454.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.28790507 - 0.9382599
Average (Standard dev.)0.00088605745 (±0.010255964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z232.960232.960232.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ470470470
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.2880.9380.001

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Supplemental data

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Sample components

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Entire : Ternary complex of the NDP-alpha-MSH activated melanocortin 4 rec...

EntireName: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 receptor (MC4R) Gs protein complex stabilized by nanobody 35
Components
  • Complex: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 receptor (MC4R) Gs protein complex stabilized by nanobody 35
    • Protein or peptide: Melanocortin receptor 4
    • Protein or peptide: NDP-alpha-MSH (other names Afamelanotide; Scenesse)
    • Protein or peptide: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • 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
    • Protein or peptide: Camelid antibody VHH fragment - nanobody 35
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 rec...

SupramoleculeName: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 receptor (MC4R) Gs protein complex stabilized by nanobody 35
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 144.6 kDa/nm

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Macromolecule #1: Melanocortin receptor 4

MacromoleculeName: Melanocortin receptor 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.69852 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDKMV NSTHRGMHTS LHLWNRSSYR LHSNASESLG KGYSDGGCYE QLFVSPEVFV TLGVISLLEN ILVIVAIAKN KNLHSPMYF FICSLAVADM LVSVSNGSET IVITLLNSTD TDAQSFTVNI DNVIDSVICS SLLASICSLL SIAVDRYFTI F YALQYHNI ...String:
DYKDDDDKMV NSTHRGMHTS LHLWNRSSYR LHSNASESLG KGYSDGGCYE QLFVSPEVFV TLGVISLLEN ILVIVAIAKN KNLHSPMYF FICSLAVADM LVSVSNGSET IVITLLNSTD TDAQSFTVNI DNVIDSVICS SLLASICSLL SIAVDRYFTI F YALQYHNI MTVKRVGIII SCIWAACTVS GILFIIYSDS SAVIICLITM FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GT GAIRQGA NMKGAITLTI LIGVFVVCWA PFFLHLIFYI SCPQNPYCVC FMSHFNLYLI LIMCNSIIDP LIYALRSQEL RKT FKEIIC CYPLGGLCDL SSRYLEVLFQ

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Macromolecule #2: NDP-alpha-MSH (other names Afamelanotide; Scenesse)

MacromoleculeName: NDP-alpha-MSH (other names Afamelanotide; Scenesse) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.632863 KDa
SequenceString:
(ACE)SYS(NLE)EH(DPN)RW GKPV(NH2)

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Macromolecule #3: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit...

MacromoleculeName: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.32616 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG DSEKATKVQD IKNNLKEAI ETIVAAMSNL VPPVELANPE NQFRVDYILS VMNVPDFDFP PEFYEHAKAL WEDEGVRACY ERSNEYQLID C AQYFLDKI ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG DSEKATKVQD IKNNLKEAI ETIVAAMSNL VPPVELANPE NQFRVDYILS VMNVPDFDFP PEFYEHAKAL WEDEGVRACY ERSNEYQLID C AQYFLDKI DVIKQADYVP SDQDLLRCRV LTSGIFETKF QVDKVNFHMF DVGGQRDERR KWIQCFNDVT AIIFVVASSS YN MVIREDN QTNRLQEALN LFKSIWNNRW LRTISVILFL NKQDLLAEKV LAGKSKIEDY FPEFARYTTP EDATPEPGED PRV TRAKYF IRDEFLRIST ASGDGRHYCY PHFTCAVDTE NIRRVFNDCR DIIQRMHLRQ YELL

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Macromolecule #4: 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: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 37.728152 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GPGSSGSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD ...String:
GPGSSGSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD TTCALWDIET GQQTTTFTGH TGDVMSLSLA PDTRLFVSGA CDASAKLWDV REGMCRQTFT GHESDINAIC FF PNGNAFA TGSDDATCRL FDLRADQELM TYSHDNIICG ITSVSFSKSG RLLLAGYDDF NCNVWDALKA DRAGVLAGHD NRV SCLGVT DDGMAVATGS WDSFLKIWN

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Macromolecule #5: 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: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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Macromolecule #6: Camelid antibody VHH fragment - nanobody 35

MacromoleculeName: Camelid antibody VHH fragment - nanobody 35 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.71432 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPE DTAVYYCARC PAPFTRDCFD VTSTTYAYRG QGTQVTVSSH HHHHH

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 115 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium chloride
0.025 mMC9H15O6PTris(2-carboxyethyl)phosphin
1.0 mMCaCl2calcium chloride
0.001 mMC78H111N21O19NDP-alpha-MSH
0.00075 %C47H88O222,2-didecylpropane-1,3-bis-beta-D-maltopyranoside
0.00025 %C56H92O25GDN101
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 5618 / Average exposure time: 40.78 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2746119
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15)
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.18) / Number images used: 221682
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

3sn6

,

6w25

)
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7piv:
Active Melanocortin-4 receptor (MC4R)- Gs protein complex bound to agonist NDP-alpha-MSH at 2.86 A resolution.

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