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- EMDB-13331: Structure of Adenylyl cyclase 9 in complex with DARPin C4 and MANT-GTP -

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Basic information

Entry
Database: EMDB / ID: EMD-13331
TitleStructure of Adenylyl cyclase 9 in complex with DARPin C4 and MANT-GTP
Map data
Sample
  • Complex: Adenylyl cyclase 9 bound to MANT-GTP
    • Complex: Adenylate cyclase 9Adenylyl cyclase
      • Protein or peptide: Adenylate cyclase 9Adenylyl cyclase
    • Complex: DARPin C4
      • Protein or peptide: DARPin C4
Function / homology
Function and homology information


Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / PKA activation / adenylate cyclase / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / adenylate cyclase-activating adrenergic receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway ...Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / PKA activation / adenylate cyclase / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / adenylate cyclase-activating adrenergic receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / in utero embryonic development / intracellular signal transduction / plasma membrane / cytosol
Similarity search - Function
Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Adenylate and Guanylate cyclase catalytic domain / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
Biological speciesBos taurus (cattle) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 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 4, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateMar 9, 2022-
Current statusMar 9, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pd8
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7pd8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13331.png

Downloads & links

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Map

FileDownload / File: emd_13331.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: 0.01 / Movie #1: 0.012
Minimum - Maximum-0.048194874 - 0.07331784
Average (Standard dev.)0.00033228414 (±0.0020681026)
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-0.0480.0730.000

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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: Adenylate cyclase 9Adenylyl cyclase
      • Protein or peptide: Adenylate cyclase 9Adenylyl cyclase
    • Complex: DARPin C4
      • Protein or peptide: DARPin C4

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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: all
Molecular weightTheoretical: 146 kDa/nm

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Supramolecule #2: Adenylate cyclase 9

SupramoleculeName: Adenylate cyclase 9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Human Embryonic Kidney Cells 293

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Supramolecule #3: DARPin C4

SupramoleculeName: DARPin C4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

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Macromolecule #2: DARPin C4

MacromoleculeName: DARPin C4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.04283 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRGSHHHHHH GSDLGKKLLE AARAGQDDEV RILMANGADV NATDDYGHTP LHLAAWFGHL EIVEVLLKAG ADVNAADWLG DTPLHLAAR IGHLEIVEVL LKHGADVNAQ DKFGKTPFDL AIDNGNEDIA EVLQKAAKLN DYKDDDDK

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 210729
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7pd8:
Structure of Adenylyl cyclase 9 in complex with DARPin C4 and MANT-GTP

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