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- EMDB-33540: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity sc... -

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Basic information

Entry
Database: EMDB / ID: EMD-33540
TitleCryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
Map dataCombined map and sharpened with Bfactor of -50 in Relion
Sample
  • Complex: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
    • Protein or peptide: DNA integrity scanning protein DisA
Keywordssecond messengers / stress response / c-di-AMP / cryo-EM / CELL CYCLE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGautam S / Vinothkumar KR / Chatterji D
Funding support India, 2 items
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, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33540.png

Downloads & links

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Map

FileDownload / File: emd_33540.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCombined map and sharpened with Bfactor of -50 in Relion
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 1.4
Minimum - Maximum-5.6700954 - 8.803793000000001
Average (Standard dev.)0.0020104963 (±0.12417459)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 470.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: One of the half maps from Cryosparc

Fileemd_33540_half_map_1.map
AnnotationOne of the half maps from Cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: One of the half maps from Cryosparc

Fileemd_33540_half_map_2.map
AnnotationOne of the half maps from Cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of the Mycobacterium smegmatis DNA integrity sc...

EntireName: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
Components
  • Complex: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
    • Protein or peptide: DNA integrity scanning protein DisA

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Supramolecule #1: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity sc...

SupramoleculeName: Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 330 KDa

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Macromolecule #1: DNA integrity scanning protein DisA

MacromoleculeName: DNA integrity scanning protein DisA / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: diadenylate cyclase
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 41.844199 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAVKSGARSG RNVVHLARPT LRETLGRLAP GTPLRDGLER ILRGRTGALI VLGYDDSVEA ICDGGFVLDV RYAPTRLREL SKMDGAVVL SSDGSRILRA NVQLVPDPSI PTDESGTRHR SAERTAIQTG YPVISVSHSM SIVTVYVAGE RHVVPDSATI L SRANQTIA ...String:
MAVKSGARSG RNVVHLARPT LRETLGRLAP GTPLRDGLER ILRGRTGALI VLGYDDSVEA ICDGGFVLDV RYAPTRLREL SKMDGAVVL SSDGSRILRA NVQLVPDPSI PTDESGTRHR SAERTAIQTG YPVISVSHSM SIVTVYVAGE RHVVPDSATI L SRANQTIA TLERYKGRLD EVSRQLSTAE IEDFVTLRDV MTVVQRLEMV RRISLEIDAD VVELGTDGRQ LKLQLDELVG DN ETARELI VRDYHANPDP PTAAQVAATL EELDSLSDSE LLDFTVLARV FGYPSTAEAQ DSAMSSRGYR AMAAIPRLQF AHV DLLVRS FGSLQNLLAA SADDLQSVDG IGSMWARHIR EGLSLLAEST IADRLAAAAL EHHHHHH

UniProtKB: DNA integrity scanning protein DisA

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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

Concentration0.1 mg/mL
BufferpH: 7.5 / Component - Concentration: 75.0 mM / Component - Formula: NaCl / Component - Name: Sodium Chloride
Details: Buffer were made freshly with 75mM NaCl and 50mM Tris (pH 7.5)
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3.5s.
DetailsThe protein sample was made on a holey carbon grid with an additional layer of thin carbon.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1587 / Average exposure time: 60.0 sec. / Average electron dose: 27.75 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130841 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 2.1 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
Startup modelType of model: OTHER / Details: Initial model was obtained with cryosparc 3.0.
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 204932
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel from AlphaFold was used as the starting point (AF-A0R564-F1-model_v2.pdb).
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 138.7
Output model

PDB-7y0d:
Cryo-EM structure of the Mycobacterium smegmatis DNA integrity scanning protein (MsDisA).

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