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- PDB-9g69: Cryo-EM structure of CdaA-DAC domain in complex with GlmM -

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Basic information

Entry
Database: PDB / ID: 9g69
TitleCryo-EM structure of CdaA-DAC domain in complex with GlmM
Components
  • Diadenylate cyclase
  • Phosphoglucosamine mutase
KeywordsPROTEIN BINDING / cyclic-di-AMP cyclace / Inhibitor / Complex
Function / homology
Function and homology information


phosphoglucosamine mutase / phosphoglucosamine mutase activity / phosphomannomutase activity / diadenylate cyclase / diadenylate cyclase activity / UDP-N-acetylglucosamine biosynthetic process / cAMP biosynthetic process / adenylate cyclase activity / peptidoglycan biosynthetic process / carbohydrate metabolic process ...phosphoglucosamine mutase / phosphoglucosamine mutase activity / phosphomannomutase activity / diadenylate cyclase / diadenylate cyclase activity / UDP-N-acetylglucosamine biosynthetic process / cAMP biosynthetic process / adenylate cyclase activity / peptidoglycan biosynthetic process / carbohydrate metabolic process / magnesium ion binding / ATP binding / plasma membrane / cytosol
Similarity search - Function
Phosphoglucosamine mutase, bacterial type / : / Diadenylate cyclase CdaA, N-terminal domain / CdaA N-terminal transmembrane domain / Diadenylate cyclase CdaA / Diadenylate cyclase / : / DNA integrity scanning protein, DisA, N-terminal / DNA integrity scanning protein, DisA, N-terminal domain superfamily / DisA bacterial checkpoint controller nucleotide-binding ...Phosphoglucosamine mutase, bacterial type / : / Diadenylate cyclase CdaA, N-terminal domain / CdaA N-terminal transmembrane domain / Diadenylate cyclase CdaA / Diadenylate cyclase / : / 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. / Alpha-D-phosphohexomutase, C-terminal / Phosphoglucomutase/phosphomannomutase, C-terminal domain / Alpha-D-phosphohexomutase superfamily / Alpha-D-phosphohexomutase, alpha/beta/alpha domain II / Alpha-D-phosphohexomutase, alpha/beta/alpha domain III / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain II / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain III / Alpha-D-phosphohexomutase, conserved site / Phosphoglucomutase and phosphomannomutase phosphoserine signature. / Alpha-D-phosphohexomutase, alpha/beta/alpha domain I / Alpha-D-phosphohexomutase, alpha/beta/alpha I/II/III / Alpha-D-phosphohexomutase, C-terminal domain superfamily / Phosphoglucomutase/phosphomannomutase, alpha/beta/alpha domain I
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Diadenylate cyclase / Phosphoglucosamine mutase
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.84 Å
AuthorsDrougkas, P. / Paulino, C. / Poolman, B.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO) Netherlands
CitationJournal: Commun Biol / Year: 2024
Title: Membrane-embedded CdaA is required for efficient synthesis of 2nd messenger cyclic di-AMP
Authors: Foster, A.J. / Li, H. / Drougkas, P. / Schuurman-Wolters, G.K. / Kate, J.T. / Paulino, C. / Poolman, B.
History
DepositionJul 18, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphoglucosamine mutase
B: Phosphoglucosamine mutase
C: Diadenylate cyclase
D: Diadenylate cyclase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,4185
Polymers161,9114
Non-polymers5071
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Phosphoglucosamine mutase


Mass: 48521.879 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: glmM, LL0424, L35068
Production host: Lactococcus lactis subsp. lactis (lactic acid bacteria)
References: UniProt: Q9CID9, phosphoglucosamine mutase
#2: Protein Diadenylate cyclase / DAC / Cyclic-di-AMP synthase / c-di-AMP synthase


Mass: 32433.725 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: dacA, llmg_0448
Production host: Lactococcus lactis subsp. lactis (lactic acid bacteria)
References: UniProt: A2RIF7, diadenylate cyclase
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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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: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2200 mMSodium ClorideNaCL1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Full-length CdaA
Specimen supportDetails: 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K
Details: 13.2 uM of GlmM and 10 mM of Mn-ATP were added prior to sample application onto the grids. Grids were blotted for 4 seconds.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 53.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5150
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
1crYOLO1.8.4particle selection
2SerialEMver4.0.6image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.9.8.91model fitting
9RELION5.0-betainitial Euler assignment
10RELION5.0-betafinal Euler assignment
12RELION5.0-beta3D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1908105
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133430 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingDetails: v3 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025051
ELECTRON MICROSCOPYf_angle_d0.4997000
ELECTRON MICROSCOPYf_dihedral_angle_d5.441027
ELECTRON MICROSCOPYf_chiral_restr0.046914
ELECTRON MICROSCOPYf_plane_restr0.0021025

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