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- EMDB-13336: focus refinement of soluble domain of adenylyl cyclase 9 in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-13336
Titlefocus refinement of soluble domain of adenylyl cyclase 9 in complex with Gs protein alpha subunit and MANT-GTP
Map data
Sample
  • Complex: Adenylyl cyclase 9 bound to MANT-GTP
    • Complex: Adenylyl cyclase 9
      • Protein or peptide: Adenylate cyclase 9
    • Complex: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsmembrane protein / adenylyl cyclase / signalling transduction. / SIGNALING PROTEIN
Function / homology
Function and homology information


Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / sensory perception of chemical stimulus / PKA activation / adenylate cyclase / mu-type opioid receptor binding / Hedgehog 'off' state / corticotropin-releasing hormone receptor 1 binding / cAMP biosynthetic process / adenylate cyclase activity ...Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / sensory perception of chemical stimulus / PKA activation / adenylate cyclase / mu-type opioid receptor binding / Hedgehog 'off' state / corticotropin-releasing hormone receptor 1 binding / cAMP biosynthetic process / adenylate cyclase activity / beta-2 adrenergic receptor binding / G alpha (z) signalling events / D1 dopamine receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / insulin-like growth factor receptor binding / adenylate cyclase activator activity / ionotropic glutamate receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / in utero embryonic development / intracellular signal transduction / GTPase activity / GTP binding / ATP binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase / G-protein alpha subunit, group S / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion ...Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase / G-protein alpha subunit, group S / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
adenylate cyclase / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsQi C / Korkhov VM
Funding support2 items
OrganizationGrant numberCountry
Swiss National Science Foundation150665
Swiss National Science Foundation176992
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of adenylyl cyclase 9 activation.
Authors: Chao Qi / Pia Lavriha / Ved Mehta / Basavraj Khanppnavar / Inayathulla Mohammed / Yong Li / Michalis Lazaratos / Jonas V Schaefer / Birgit Dreier / Andreas Plückthun / Ana-Nicoleta Bondar / ...Authors: Chao Qi / Pia Lavriha / Ved Mehta / Basavraj Khanppnavar / Inayathulla Mohammed / Yong Li / Michalis Lazaratos / Jonas V Schaefer / Birgit Dreier / Andreas Plückthun / Ana-Nicoleta Bondar / Carmen W Dessauer / Volodymyr M Korkhov /
Abstract: Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the ...Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.
History
DepositionAug 5, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pdf
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13336.png

Downloads & links

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Map

FileDownload / File: emd_13336.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 300 pix.
= 243. Å		0.81 Å/pix.
x 300 pix.
= 243. Å		0.81 Å/pix.
x 300 pix.
= 243. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.04818577 - 0.07588037
Average (Standard dev.)0.000113403024 (±0.0017004699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 243.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z243.000243.000243.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0480.0760.000

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Supplemental data

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Sample components

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Entire : Adenylyl cyclase 9 bound to MANT-GTP

EntireName: Adenylyl cyclase 9 bound to MANT-GTP
Components
  • Complex: Adenylyl cyclase 9 bound to MANT-GTP
    • Complex: Adenylyl cyclase 9
      • Protein or peptide: Adenylate cyclase 9
    • Complex: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Adenylyl cyclase 9 bound to MANT-GTP

SupramoleculeName: Adenylyl cyclase 9 bound to MANT-GTP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 146 kDa/nm

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Supramolecule #2: Adenylyl cyclase 9

SupramoleculeName: Adenylyl cyclase 9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)

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Supramolecule #3: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

SupramoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Adenylate cyclase 9

MacromoleculeName: Adenylate cyclase 9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 151.142875 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASPPHQQLL QHHSTEVSCD SSGDSNSVRV RINPKQPSSN SHPKHCKYSI SSSCSSSGDS GGVPRRMGAG GRLRRRKKLP QLFERASSR WWDPKFDSVN LEEACMERCF PQTQRRFRYA LFYIGFACLL WSIYFGVHMK SKLIVMVAPA LCFLVVCVGF F LFTFTKLY ...String:
MASPPHQQLL QHHSTEVSCD SSGDSNSVRV RINPKQPSSN SHPKHCKYSI SSSCSSSGDS GGVPRRMGAG GRLRRRKKLP QLFERASSR WWDPKFDSVN LEEACMERCF PQTQRRFRYA LFYIGFACLL WSIYFGVHMK SKLIVMVAPA LCFLVVCVGF F LFTFTKLY ARHYVWTSLV LTLLVFALTL AAQFQVLTPL SGRVDNFNHT RAARPTDTCL SQVGSFSMCI EVLFLLYTVM HL PLYLSLI LGVAYSVLFE TFGYHFQDEA CFASPGAEAL HWELLSRALL HLCIHAIGIH LFIMSQVRSR STFLKVGQSI MHG KDLEVE KALKERMIHS VMPRIIADDL MKQGDEESEN SVKRHATSSP KNRKKKSSIQ KAPIAFRPFK MQQIEEVSIL FADI VGFTK MSANKSAHAL VGLLNDLFGR FDRLCEETKC EKISTLGDCY YCVAGCPEPR ADHAYCCIEM GLGMIRAIEQ FCQEK KEMV NMRVGVHTGT VLCGILGMRR FKFDVWSNDV NLANLMEQLG VAGKVHISEA TAKYLDDRYE MEDGKVTERL GQSVVA DQL KGLKTYLIAG QRAKESHCSC SEALLSGFEV LDGSRVSSGP RGQGTASPGS VSDLAQTVKT FDNLKTCPSC GITFTPK PE AGAEGGAVQN GCQEEPKNSA KASGGPSSKT QNGLLSPPPE EKLTNSQTSL CEILQEKGRW AGVSLDQSAL LPLRFKNI R EKTDAHFVDV IKEDSLMKDY FFKPPINQFS LNFLDPELER AYRTSYQEEV VKSSPVRTFA SATFSSLLDV LLSTTVFLI LSITCFLRYG AASTPPPPAA LAVFGAALLL EILSLVVSVR MVFFLEDVMT CTKRLLEWIA GWLPRHFIGA ILVSLPALAV YSHVTSEFE TNIHSTMFTG SAVLTAVVQY CNFCQLSSWM RSSLATVVGA GPLLLLLYVS LCPDSSTVIS HLDAVQNFSS T RKLCNASL PHDGRSPASL IGQEVILVFF LLLLLVWFLN REFEVSYRLH YHGDVEADLH RTKIQSMRDQ ADWLLRNIIP YH VAEQLKV SQTYSKNHDS GGVIFASIVN FSEFYEENYE GGKECYRVLN ELIGDFDELL SKPDYSSIEK IKTIGATYMA ASG LNATQC RDGSHPQEHL QILFEFAKEM MRVVDDFNNN MLWFNFKLRV GFNHGPLTAG VIGTTKLLYD IWGDTVNIAS RMDT TGVEC RIQVSEESYR VLSKMGYEFD YRGTVNVKGK GQMKTYLYPK CTDSGLVPQH QLSISPDIRV QVDGSIGRSP TDEIA SLVP SVQNPDQVPP GSENNAQTRD AHPSAKRPWK EPVRAEERCR FGKAIEKSDC EEVGMEEANE LTKLNVSERA

UniProtKB: adenylate cyclase

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Macromolecule #2: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 47.003801 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEK ATKVQDIKNN LKEAIETIVA AMSNLVPPVE LANPENQFRV DYILSVMNVP DFDFPPEFYE HAKALWEDEG V RACYERSN ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEK ATKVQDIKNN LKEAIETIVA AMSNLVPPVE LANPENQFRV DYILSVMNVP DFDFPPEFYE HAKALWEDEG V RACYERSN EYQLIDCAQY FLDKIDVIKQ DDYVPSDQDL LRCRVLTSGI FETKFQVDKV NFHMFDVGGQ RDERRKWIQC FN DVTAIIF VVASSSYNMV IREDNQTNRL QEALNLFKSI WNNRWLRTIS VILFLNKQDL LAEKVLAGKS KIEDYFPEFA RYT TPEDAT PEPGEDPRVT RAKYFIRDEF LRISTASGDG RHYCYPHFTC AVDTENIRRV FNDCRDIIQR MHLRQYELLG GHHH HHHHH

UniProtKB: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Macromolecule #3: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GSP
Molecular weightTheoretical: 539.246 Da
Chemical component information

ChemComp-GSP:
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 157200
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7pdf:
focus refinement of soluble domain of adenylyl cyclase 9 in complex with Gs protein alpha subunit and MANT-GTP

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