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- PDB-6r4o: Structure of a truncated adenylyl cyclase bound to MANT-GTP, fors... -

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Basic information

Entry
Database: PDB / ID: 6r4o
TitleStructure of a truncated adenylyl cyclase bound to MANT-GTP, forskolin and an activated stimulatory Galphas protein
Components
  • Adenylate cyclase 9
  • Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
KeywordsMEMBRANE PROTEIN / Adenylyl cyclase / G protein / MANT-GTP / forskolin
Function / homology
Function and homology information


Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / sensory perception of chemical stimulus / PKA activation / adenylate cyclase / Hedgehog 'off' state / mu-type opioid receptor binding / 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 / Hedgehog 'off' state / mu-type opioid receptor binding / 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 / ionotropic glutamate receptor binding / adenylate cyclase activator activity / 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 / ciliary basal body / GTPase activity / GTP binding / ATP binding / metal ion binding / plasma membrane / cytoplasm / cytosol
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
FORSKOLIN / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / : / Chem-ONM / Adenylate cyclase type 9 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesBos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsQi, C. / Sorrentino, S. / Medalia, O. / Korkhov, V.M.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation150665 Switzerland
Citation
Journal: Science / Year: 2019
Title: The structure of a membrane adenylyl cyclase bound to an activated stimulatory G protein.
Authors: Chao Qi / Simona Sorrentino / Ohad Medalia / Volodymyr M Korkhov /
Abstract: Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. ...Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. Signals received by the G protein-coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo-electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.
#1: Journal: To Be Published
Title: The structure of a membrane adenylyl cyclase bound to an activated stimulatory G protein
Authors: Qi, C. / Sorrentino, S. / Medalia, O. / Korkhov, V.M.
History
DepositionMar 22, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / em_image_scans / Item: _em_3d_fitting.target_criteria
Revision 1.2May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Adenylate cyclase 9
B: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,6968
Polymers217,9562
Non-polymers1,7406
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5260 Å2
ΔGint-49 kcal/mol
Surface area63010 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Adenylate cyclase 9 / Adenylyl CYclase family member (Acy-1)-like


Mass: 170951.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Bovine AC9, residues 1-1250, tagged with 3C-YFP-twinStrep tag
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: ADCY9, BOS_22626 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: E1BM79
#2: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 47003.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GNAS, GNAS1 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P04896

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Non-polymers , 5 types, 6 molecules

#3: Chemical ChemComp-ONM / 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE


Mass: 656.328 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H23N6O15P3
#4: Chemical ChemComp-FOK / FORSKOLIN


Mass: 410.501 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H34O7
#5: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#6: Chemical ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3S
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-GTP, forskolin and GalphaS proteinCOMPLEX#1-#20MULTIPLE SOURCES
2Adenylyl cyclase AC9COMPLEX#11RECOMBINANT
3GalphaS proteinCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bos taurus (domestic cattle)9913
33Bos taurus (domestic cattle)9913
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606HEK293F
33Trichoplusia ni (cabbage looper)7111High Five
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10758
Details: Two datasets were merged using two different microscopes, using pixel size of 0.831 A/pix (4453 micrographs; 60 e-/A2) and 0.8544 A/pix (6305 micrographs; 45 e-/A2). Final dataset was scaled to 0.8544 A/pix.

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9PHENIX1.11.1model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13cisTEM1.0.03D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1107881
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142147 / Symmetry type: POINT
Atomic model buildingB value: 110.92 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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