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- PDB-7yzi: Structure of Mycobacterium tuberculosis adenylyl cyclase Rv1625c / Cya -

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Basic information

Entry
Database: PDB / ID: 7yzi
TitleStructure of Mycobacterium tuberculosis adenylyl cyclase Rv1625c / Cya
Components
  • Adenylate cyclase
  • Nanobody Nb4
KeywordsMEMBRANE PROTEIN / Adenylyl cyclase / cyclic adenosine monophosphate / signal transduction / nanobody
Function / homology
Function and homology information


receptor guanylyl cyclase signaling pathway / peptide receptor activity / cGMP biosynthetic process / guanylate cyclase activity / adenylate cyclase / cAMP biosynthetic process / adenylate cyclase activity / manganese ion binding / intracellular signal transduction / magnesium ion binding ...receptor guanylyl cyclase signaling pathway / peptide receptor activity / cGMP biosynthetic process / guanylate cyclase activity / adenylate cyclase / cAMP biosynthetic process / adenylate cyclase activity / manganese ion binding / intracellular signal transduction / magnesium ion binding / ATP binding / plasma membrane
Similarity search - Function
: / MASE7 / : / 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
Similarity search - Domain/homology
: / Chem-ONM / Adenylate cyclase
Similarity search - Component
Biological speciesMycobacterium tuberculosis '98-R604 INH-RIF-EM' (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.83 Å
AuthorsMehta, V. / Khanppnavar, B. / Korkhov, V.M.
Funding support Switzerland, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation150665 Switzerland
Swiss National Science Foundation176992 Switzerland
Swiss National Science Foundation184951 Switzerland
CitationJournal: Elife / Year: 2022
Title: Structure of Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.
Authors: Ved Mehta / Basavraj Khanppnavar / Dina Schuster / Ilayda Kantarci / Irene Vercellino / Angela Kosturanova / Tarun Iype / Sasa Stefanic / Paola Picotti / Volodymyr M Korkhov /
Abstract: adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular ... adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.
History
DepositionFeb 20, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Adenylate cyclase
B: Adenylate cyclase
C: Nanobody Nb4
D: Nanobody Nb4
E: Nanobody Nb4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,61911
Polymers142,0875
Non-polymers1,5326
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Adenylate cyclase / ATP pyrophosphate-lyase / Adenylyl cyclase


Mass: 50386.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis '98-R604 INH-RIF-EM' (bacteria)
Gene: cya, Rv1625c, MTCY01B2.17c / Production host: Escherichia coli (E. coli) / References: UniProt: P9WQ35, adenylate cyclase
#2: Antibody Nanobody Nb4


Mass: 13771.179 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-ONM / 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE


Mass: 656.328 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H23N6O15P3
#4: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestN

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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
1A complex of Rv1625c / Cya homodimer bound to nano body Nb4COMPLEX#1-#20RECOMBINANT
2Adenylate cyclaseCOMPLEX#11RECOMBINANT
3Nanobody Nb4COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mycobacterium tuberculosis '98-R604 INH-RIF-EM' (bacteria)555461
33Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5 / Details: 50 mM Tris pH 7.5, 200 mM NaCl, 0.1 % digitonin
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 646042 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.018690
ELECTRON MICROSCOPYf_angle_d1.8111871
ELECTRON MICROSCOPYf_dihedral_angle_d9.8421220
ELECTRON MICROSCOPYf_chiral_restr0.2021367
ELECTRON MICROSCOPYf_plane_restr0.0331513

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