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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, conserved site / Phosphoglucomutase and phosphomannomutase phosphoserine signature. / 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, 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 second messenger cyclic di-AMP.
Authors: Alexander J Foster / Haoyang Li / Panagiotis Drougkas / Gea K Schuurman-Wolters / Joeri Ten Kate / Cristina Paulino / Bert Poolman /
Abstract: Cyclic di-adenylate monophosphate (cyclic di-AMP) is an important second messenger in microorganisms. Cyclic di-AMP regulates bacterial cell volume and turgor via control of potassium and compatible ...Cyclic di-adenylate monophosphate (cyclic di-AMP) is an important second messenger in microorganisms. Cyclic di-AMP regulates bacterial cell volume and turgor via control of potassium and compatible solute transport but is also involved in many other processes, including the activation of the metazoan innate immune response to bacterial infections. We compare the activity of full-length membrane-embedded CdaA, the enzyme that synthesizes cyclic di-AMP, with the water-soluble catalytic domain CdaA-DAC. Purified CdaA from L. lactis was studied in the detergent-solubilized state, and in lipid nanodiscs and vesicles. We show that CdaA is tetrameric and the membrane-bound complex has more than 2-orders of magnitude higher activity than soluble CdaA-DAC. CdaA activity increases with pH but does not strongly depend on the salt or lipid content, factors that are crucial for the control of osmoregulatory transporters. Cryo-EM and in-silico structure prediction of CdaA show that the two DAC dimers engage in a head-to-head interaction, leading to cyclic-di-AMP formation. The inhibitor phosphoglucomutase prevents this active conformation. We observe dynamic flexibility between the catalytic and membrane domains, even in the presence of ATP or non-hydrolyzable substrate ApCpp. This is the first comprehensive functional and structural characterization of a full-length cyclic di-AMP-specific cyclase.
History
DepositionJul 18, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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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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