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- EMDB-4721: Structure of a truncated adenylyl cyclase bound to MANT-GTP, fors... -

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Basic information

Entry
Database: EMDB / ID: EMD-4721
TitleStructure of a truncated adenylyl cyclase bound to MANT-GTP, forskolin and an activated stimulatory Galphas protein
Map dataTruncated AC9 (AC9-1250) bound to MANT-GTP, forskolin and GalphaS
Sample
  • Complex: Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-GTP, forskolin and GalphaS protein
    • Complex: Adenylyl cyclase AC9
      • Protein or peptide: Adenylate cyclase 9Adenylyl cyclase
    • Complex: GalphaS protein
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: FORSKOLIN
  • Ligand: MANGANESE (II) ION
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION
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 / G alpha (z) signalling events / beta-2 adrenergic receptor binding / D1 dopamine receptor binding / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / ionotropic glutamate receptor binding / insulin-like growth factor receptor binding / adenylate cyclase activator activity / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of GTPase activity / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex / in utero embryonic development / intracellular signal transduction / GTPase activity / GTP binding / metal ion binding / 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 / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit ...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 / 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 / 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: 4.2 Å
AuthorsQi C / Sorrentino S / Medalia O / Korkhov VM
Funding support Switzerland, 1 items
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 VM
History
DepositionMar 22, 2019-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 8, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6r4o
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4721.png

Downloads & links

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Map

FileDownload / File: emd_4721.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTruncated AC9 (AC9-1250) bound to MANT-GTP, forskolin and GalphaS
Voxel sizeX=Y=Z: 0.8544 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.8
Minimum - Maximum-5.1731133 - 11.672428
Average (Standard dev.)-0.0047368403 (±0.43477216)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 341.75998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.85440.85440.8544
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z341.760341.760341.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-5.17311.672-0.005

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Supplemental data

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Sample components

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Entire : Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-...

EntireName: Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-GTP, forskolin and GalphaS protein
Components
  • Complex: Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-GTP, forskolin and GalphaS protein
    • Complex: Adenylyl cyclase AC9
      • Protein or peptide: Adenylate cyclase 9Adenylyl cyclase
    • Complex: GalphaS protein
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: FORSKOLIN
  • Ligand: MANGANESE (II) ION
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-...

SupramoleculeName: Adenylyl cyclase AC9 (truncation, residues 1-1250) bound to MANT-GTP, forskolin and GalphaS protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Adenylyl cyclase AC9

SupramoleculeName: Adenylyl cyclase AC9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Supramolecule #3: GalphaS protein

SupramoleculeName: GalphaS protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High Five

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Macromolecule #1: Adenylate cyclase 9

MacromoleculeName: Adenylate cyclase 9 / type: protein_or_peptide / ID: 1
Details: Bovine AC9, residues 1-1250, tagged with 3C-YFP-twinStrep tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 170.951953 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 CTDSGAAALE VLFQGPGGVS KGEELFTGVV PILVE LDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV TTFGYGLQCF ARYPDHMKQH DFFKSAMPEG YVQERT IFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQ LA DHYQQNTPIG DGPVLLPDNH YLSYQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKA ASAWSHPQFE KGGGSGGG S GGSAWSHPQF EK

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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

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Macromolecule #3: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ONM
Molecular weightTheoretical: 656.328 Da
Chemical component information

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

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Macromolecule #4: FORSKOLIN

MacromoleculeName: FORSKOLIN / type: ligand / ID: 4 / Number of copies: 1 / Formula: FOK
Molecular weightTheoretical: 410.501 Da
Chemical component information

ChemComp-FOK:
FORSKOLIN / Forskolin

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Macromolecule #5: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #6: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 6 / 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 #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / 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

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 10758 / Average electron dose: 60.0 e/Å2
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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1107881
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: AC9-GalphaS (EMD-4719) was used as initial model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 142147
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 110.92 / Target criteria: Cross-correlation coefficient
Output model

PDB-6r4o:
Structure of a truncated adenylyl cyclase bound to MANT-GTP, forskolin and an activated stimulatory Galphas protein

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