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- PDB-7y0d: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity sc... -

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Basic information

Entry
Database: PDB / ID: 7y0d
TitleCryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
ComponentsDNA integrity scanning protein DisA
KeywordsCELL CYCLE / second messengers / stress response / c-di-AMP / cryo-EM
Function / homology
Function and homology information


: / diadenylate cyclase activity / diadenylate cyclase / DNA repair / DNA binding / ATP binding
Similarity search - Function
DNA integrity scanning, DisA, linker region / DNA integrity scanning protein, DisA / DisA, linker domain superfamily / DisA bacterial checkpoint controller linker region / DNA integrity scanning protein, DisA, N-terminal / DNA integrity scanning protein, DisA, N-terminal domain superfamily / DisA bacterial checkpoint controller nucleotide-binding / Diadenylate cyclase (DAC) domain profile. / DisA/LigA, helix-hairpin-helix motif / Helix-hairpin-helix motif / RuvA domain 2-like
Similarity search - Domain/homology
DNA integrity scanning protein DisA
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGautam, S. / Vinothkumar, K.R. / Chatterji, D.
Funding support India, 2items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB)RJN-094/2017 India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: Protein Sci / Year: 2023
Title: Regulatory mechanisms of c-di-AMP synthase from Mycobacterium smegmatis revealed by a structure: Function analysis.
Authors: Sudhanshu Gautam / Avisek Mahapa / Lahari Yeramala / Apoorv Gandhi / Sushma Krishnan / Vinothkumar Kutti R / Dipankar Chatterji /
Abstract: Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a ...Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.
History
DepositionJun 4, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 1, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA integrity scanning protein DisA
B: DNA integrity scanning protein DisA
C: DNA integrity scanning protein DisA
D: DNA integrity scanning protein DisA
E: DNA integrity scanning protein DisA
F: DNA integrity scanning protein DisA
G: DNA integrity scanning protein DisA
H: DNA integrity scanning protein DisA


Theoretical massNumber of molelcules
Total (without water)334,7548
Polymers334,7548
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, Size-exclusion chromatography with Multi Angle Light Scattering is used to validate octameric nature of MsDisA. TEM analysis also supports the octameric assembly.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DNA integrity scanning protein DisA / Cyclic-di-AMP synthase / c-di-AMP synthase / Diadenylate cyclase


Mass: 41844.199 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: disA, MSMEG_6080, MSMEI_5920 / Plasmid: pET28A / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0R564, diadenylate cyclase

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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

ComponentName: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.33 MDa / Experimental value: YES
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: Buffer were made freshly with 75mM NaCl and 50mM Tris (pH 7.5)
Buffer componentConc.: 75 mM / Name: Sodium Chloride / Formula: NaCl
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The protein sample was made on a holey carbon grid with an additional layer of thin carbon.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Details: blotted for 3.5s

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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 / Nominal magnification: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 3300 nm / Nominal defocus min: 2100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 27.75 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1587
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
7Coot0.9.5model fitting
8UCSF Chimera1.15model fitting
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
14PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 973755
Details: Template Picker from cryoSparc 3.0 were used to pick total particles in the first extraction. Three rounds of reference-free 2d classification were carried to remove bad particles followed by NU-refinement.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204932 / Algorithm: BACK PROJECTION / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingB value: 138.7 / Protocol: OTHER / Space: REAL
Details: Model from AlphaFold was used as the starting point (AF-A0R564-F1-model_v2.pdb).

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