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- PDB-7yz9: Structure of catalytic domain of Rv1625c bound to nanobody NB4 -

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Basic information

Entry
Database: PDB / ID: 7yz9
TitleStructure of catalytic domain of Rv1625c bound to nanobody NB4
Components
  • Adenylate cyclaseAdenylyl cyclase
  • nanobody NB4
KeywordsSIGNALING PROTEIN / Membrane Adenylyl Cyclase / Nanobody / Complex / Mycobacterium Tuberculosis
Function / homology
Function and homology information


receptor guanylyl cyclase signaling pathway / peptide receptor activity / guanylate cyclase activity / cGMP biosynthetic process / 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 / guanylate cyclase activity / cGMP biosynthetic process / 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 / Adenylyl cyclase class-3/4/guanylyl cyclase / Adenylate and Guanylate cyclase catalytic domain / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
: / Chem-ONM / Adenylate cyclase
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.97 Å
AuthorsKhanppnavar, B. / Mehta, V.J. / Iype, T. / 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 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Adenylate cyclase
B: nanobody NB4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,9798
Polymers41,9742
Non-polymers1,0056
Water2,792155
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2860 Å2
ΔGint-24 kcal/mol
Surface area14500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.793, 94.793, 119.655
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Space group name HallP652(x,y,z+1/12)
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+1/3
#8: -x,-y,z+1/2
#9: y,x,-z+2/3
#10: -y,-x,-z+1/6
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+5/6
Components on special symmetry positions
IDModelComponents
11A-421-

HOH

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Components

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Protein / Antibody , 2 types, 2 molecules AB

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


Mass: 28202.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 161 molecules

#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-ONM / 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE


Mass: 656.328 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H23N6O15P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 155 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.35 Å3/Da / Density % sol: 47.67 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 0.1 M Na-acetate pH 5.5, 0.02 M CaCl2, 30% MPD / PH range: 5.0-6.5

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Data collection

DiffractionMean temperature: 90 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 0.999879 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 1, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.999879 Å / Relative weight: 1
ReflectionResolution: 1.97→47.4 Å / Num. obs: 774280 / % possible obs: 93.96 % / Redundancy: 35.9 % / Biso Wilson estimate: 44.58 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.09 / Net I/σ(I): 25.4
Reflection shellResolution: 1.973→2.044 Å / Redundancy: 22.8 % / Rmerge(I) obs: 1.587 / Mean I/σ(I) obs: 1.5 / Num. unique obs: 1492 / CC1/2: 0.685 / % possible all: 66.85

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
PHENIX1.17.1_3660refinement
iMOSFLMdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4P2F

4p2f


Resolution: 1.97→44.07 Å / SU ML: 0.272 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 27.6644
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2438 1978 4.96 %
Rwork0.2056 37862 -
obs0.2076 39840 94.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.38 Å2
Refinement stepCycle: LAST / Resolution: 1.97→44.07 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2266 0 57 155 2478
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01212431
X-RAY DIFFRACTIONf_angle_d1.16353312
X-RAY DIFFRACTIONf_chiral_restr0.0569360
X-RAY DIFFRACTIONf_plane_restr0.0045430
X-RAY DIFFRACTIONf_dihedral_angle_d20.9456871
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.97-2.020.3506950.31911866X-RAY DIFFRACTION65.28
2.02-2.080.3261120.28572147X-RAY DIFFRACTION74.48
2.08-2.140.37731250.28132402X-RAY DIFFRACTION83.62
2.14-2.210.26351450.26552745X-RAY DIFFRACTION95.1
2.21-2.290.30651500.2632819X-RAY DIFFRACTION99.4
2.29-2.380.3141510.2612882X-RAY DIFFRACTION99.97
2.38-2.490.30681530.24472872X-RAY DIFFRACTION100
2.49-2.620.25931510.24882864X-RAY DIFFRACTION100
2.62-2.780.26081470.2372902X-RAY DIFFRACTION100
2.78-30.30141520.23322865X-RAY DIFFRACTION100
3-3.30.23251570.20572861X-RAY DIFFRACTION100
3.3-3.770.2021470.18162871X-RAY DIFFRACTION100
3.77-4.750.18631470.16162885X-RAY DIFFRACTION99.9
4.75-44.070.261460.19442881X-RAY DIFFRACTION99.97

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