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- EMDB-16254: Focused refinement of transmembrane domain of Adenylyl cyclase 8 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-16254
TitleFocused refinement of transmembrane domain of Adenylyl cyclase 8 bound to stimulatory G-protein, Ca2+/Calmodulin, Forskolin and MANT-GTP
Map data
Sample
  • Complex: Focused refinement of soluble domain of Adenylyl cyclase 8 bound to stimulatory G protein, Forskolin, and MANT-GTP
    • Complex: Adenylyl cyclase 8
      • Protein or peptide: Adenylyl cyclase 8
    • Complex: G protein alpha S
      • Protein or peptide: G protein alpha S
KeywordsAdenylyl Cyclase / cAMP signaling / G proteins / Calmodulin / MEMBRANE PROTEIN
Function / homology
Function and homology information


cellular response to morphine / Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / positive regulation of long-term synaptic depression / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / PKA activation / neuroinflammatory response / adenylate cyclase ...cellular response to morphine / Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / positive regulation of long-term synaptic depression / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / PKA activation / neuroinflammatory response / adenylate cyclase / positive regulation of synaptic plasticity / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / neuronal cell body membrane / cellular response to glucagon stimulus / presynaptic active zone / positive regulation of insulin secretion involved in cellular response to glucose stimulus / excitatory synapse / long-term memory / regulation of cytosolic calcium ion concentration / hippocampal mossy fiber to CA3 synapse / locomotory behavior / positive regulation of long-term synaptic potentiation / Schaffer collateral - CA1 synapse / adenylate cyclase-activating G protein-coupled receptor signaling pathway / presynaptic membrane / basolateral plasma membrane / postsynaptic density / intracellular signal transduction / apical plasma membrane / axon / glutamatergic synapse / dendrite / ATP binding / metal ion binding
Similarity search - Function
Adenylate cyclase, conserved domain / Adenylate cyclase / Adenylate cyclase, N-terminal / Adenylate cyclase, conserved domain / Adenylyl cyclase N-terminal extracellular and transmembrane region / 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 ...Adenylate cyclase, conserved domain / Adenylate cyclase / Adenylate cyclase, N-terminal / Adenylate cyclase, conserved domain / Adenylyl cyclase N-terminal extracellular and transmembrane region / 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
Adenylate cyclase type 8
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.13 Å
AuthorsKhanppnavar B / Korkhov VM / Mehta V
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation198545 Switzerland
Swiss National Science Foundation184951 Switzerland
CitationJournal: EMBO Rep / Year: 2024
Title: Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.
Authors: Basavraj Khanppnavar / Dina Schuster / Pia Lavriha / Federico Uliana / Merve Özel / Ved Mehta / Alexander Leitner / Paola Picotti / Volodymyr M Korkhov /
Abstract: Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca signalling. Despite the importance of AC8 in physiology, the ...Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
History
DepositionDec 1, 2022-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16254.png

Downloads & links

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Map

FileDownload / File: emd_16254.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 420 pix.
= 277.2 Å		0.66 Å/pix.
x 420 pix.
= 277.2 Å		0.66 Å/pix.
x 420 pix.
= 277.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.02894622 - 0.043500073
Average (Standard dev.)0.000025960231 (±0.0005531043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 277.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half2

Fileemd_16254_half_map_1.map
Annotationhalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half1

Fileemd_16254_half_map_2.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Focused refinement of soluble domain of Adenylyl cyclase 8 bound ...

EntireName: Focused refinement of soluble domain of Adenylyl cyclase 8 bound to stimulatory G protein, Forskolin, and MANT-GTP
Components
  • Complex: Focused refinement of soluble domain of Adenylyl cyclase 8 bound to stimulatory G protein, Forskolin, and MANT-GTP
    • Complex: Adenylyl cyclase 8
      • Protein or peptide: Adenylyl cyclase 8
    • Complex: G protein alpha S
      • Protein or peptide: G protein alpha S

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Supramolecule #1: Focused refinement of soluble domain of Adenylyl cyclase 8 bound ...

SupramoleculeName: Focused refinement of soluble domain of Adenylyl cyclase 8 bound to stimulatory G protein, Forskolin, and MANT-GTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Adenylyl cyclase 8

SupramoleculeName: Adenylyl cyclase 8 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)

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Supramolecule #3: G protein alpha S

SupramoleculeName: G protein alpha S / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Adenylyl cyclase 8

MacromoleculeName: Adenylyl cyclase 8 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: adenylate cyclase
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MELSDVRCLS GSEELYTIHP TPPAGDSGSG SRPQRLLWQT AVRHITEQRF IHGHRGGGGG GGNGSSSKA SDPGGGGPNH HHASQLSGDS ALPLYALGPG ERAHGTGGPK VFPERSGSGS A SGSGGGGD LGFLHLDCAP SNSDFFLNGG YSYRGVIFPT LRNSFKSRDL ...String:
MELSDVRCLS GSEELYTIHP TPPAGDSGSG SRPQRLLWQT AVRHITEQRF IHGHRGGGGG GGNGSSSKA SDPGGGGPNH HHASQLSGDS ALPLYALGPG ERAHGTGGPK VFPERSGSGS A SGSGGGGD LGFLHLDCAP SNSDFFLNGG YSYRGVIFPT LRNSFKSRDL ERLYQRYFLG QR RKSEVVM NVLDVLTKLT LLVLHLSLAS APMDPLKGIL LGFFTGIEVV ICALVVVRKD TTS HTYLQY SGVVTWVAMT TQILAAGLGY GLLGDGIGYV LFTLFATYSM LPLPLTWAIL AGLG TSLMQ VVLQAVIPRL AVISINQVVA QAVLFMCMNT AGIFISYLSD RAQRQAFLET RRCVE ARLR LETENQRQER LVLSVLPRFV VLEMINDMTN VEDEHLQHQF HRIYIHRYEN VSILFA DVK GFTNLSTTLS AQELVRMLNE LFARFDRLAH EHHCLRIKIL GDCYYCVSGL PEPRQDH AH CCVEMGLSMI KTIRYVRSRT KHDVDMRIGI HSGSVLCGVL GLRKWQFDVW SWDVDIAN K LESGGIPGRI HISKATLDCL NGDYNVEEGH GKERNEFLRK HNIETYLIKQ PEESLLSLP EDIVKESVSS SDRRNSGATF TEGSWSPELP FDNIVGKQNT LAALTRNSIN LLPNHLAQAL HVQSGPEEI NKRIEHTIDL RSGDKLRREH IKPFSLMFKD SSLEHKYSQM RDEVFKSNLV C AFIVLLFI TAIQSLLPSS RVMPMAIQFS ILIMLHSALV LITTAEDYKC LPLVLRKTCC WI NETYLAR NVIIFASILI NFLGAILNIL WCDFDKSIPL KNLTFNSSAV FTDICSYPEY FVF TGVLAM VTCAVFLRLN SVLKLAVLLI MIAIYALLTE TIYAGLFLRY DNLNHSGEDF LGTK EASLL LMAMFLLAVF YHGQQLEYTA RLDFLWRVQA KEEINEMKEL REHNENMLRN ILPSH VARH FLEKDRDNEE LYSQSYDAVG VMFASIPGFA DFYSQTEMNN QGVECLRLLN EIIADF DEL LGEDRFQDIE KIKTIGSTYM AVSGLSPEKQ QCEDKWGHLC ALADFSLALT ESIQEIN KH SFNNFELRIG ISHGSVVAGV IGAKKPQYDI WGKTVNLASR MDSTGVSGRI QVPEETYL I LKDQGFAFDY RGEIYVKGIS EQEGKIKTYF LLGRVQPNPF ILPPRRLPGQ YSLAAVVLG LVQSLNRQRQ KQLLNENNNT GIIKGHYNRR TLLTPSGPEP GAPAEGADKP ELP

UniProtKB: Adenylate cyclase type 8

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Macromolecule #2: G protein alpha S

MacromoleculeName: G protein alpha S / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV RACYERSNEY ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV RACYERSNEY QLIDCAQYFL DKIDVIKQDD YVPSDQDLLR CRVLTSGIFE TKFQVDKVNF HMFDVGGQRD ERRKWIQCFN DVTAIIFVVA SSSYNMVIRE DNQTNRLQEA LNLFKSIWNN RWLRTISVIL FLNKQDLLAE KVLAGKSKIE DYFPEFARYT TPEDATPEPG EDPRVTRAKY FIRDEFLRIS TASGDGRHYC YPHFTCAVDT ENIRRVFNDC RDIIQRMHLR QYELLGGHHH HHHHH

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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 / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab-initial 3D model reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 77575
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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