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- EMDB-51096: Cryo-EM structure of CdaA-DAC domain in complex with GlmM -

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Basic information

Entry
Database: EMDB / ID: EMD-51096
TitleCryo-EM structure of CdaA-DAC domain in complex with GlmM
Map data
Sample
  • Complex: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP
    • Protein or peptide: Phosphoglucosamine mutase
    • Protein or peptide: Diadenylate cyclase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Keywordscyclic-di-AMP cyclace / Inhibitor / Complex / PROTEIN BINDING
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 / Alpha-D-phosphohexomutase, C-terminal / : / DNA integrity scanning protein, DisA, N-terminal / DNA integrity scanning protein, DisA, N-terminal domain superfamily ...Phosphoglucosamine mutase, bacterial type / : / Diadenylate cyclase CdaA, N-terminal domain / CdaA N-terminal transmembrane domain / Diadenylate cyclase CdaA / Diadenylate cyclase / Alpha-D-phosphohexomutase, C-terminal / : / 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. / 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
Diadenylate cyclase / Phosphoglucosamine mutase
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.84 Å
AuthorsDrougkas P / Paulino C / Poolman B
Funding support Netherlands, 1 items
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, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51096.png

Downloads & links

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Map

FileDownload / File: emd_51096.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0355
Minimum - Maximum-0.18344148 - 0.25503576
Average (Standard dev.)0.00026166305 (±0.006656628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51096_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_51096_half_map_1.map
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Half map: #1

Fileemd_51096_half_map_2.map
Projections & Slices
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Sample components

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Entire : Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP

EntireName: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP
Components
  • Complex: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP
    • Protein or peptide: Phosphoglucosamine mutase
    • Protein or peptide: Diadenylate cyclase
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP

SupramoleculeName: Diadenylate cyclase CdaA, Phosphoglucosamine mutase GlmM, ATP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)

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Macromolecule #1: Phosphoglucosamine mutase

MacromoleculeName: Phosphoglucosamine mutase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: phosphoglucosamine mutase
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 48.521879 KDa
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MGKYFGTDGV RGEANVELTP EMAFKLGRFG GYVLSQHELG TPKVYVGRDT RISGQMLASS LISGLLSVGI EVYDLGVIAT PGVAYLVKK DGASAGVMIS ASHNPALDNG IKFFGGDGYK LEDEKELEIE ALIDAEEDTL PRPSAQGLGM LHDYIEGVRK Y QAFLKTTA ...String:
MGKYFGTDGV RGEANVELTP EMAFKLGRFG GYVLSQHELG TPKVYVGRDT RISGQMLASS LISGLLSVGI EVYDLGVIAT PGVAYLVKK DGASAGVMIS ASHNPALDNG IKFFGGDGYK LEDEKELEIE ALIDAEEDTL PRPSAQGLGM LHDYIEGVRK Y QAFLKTTA EGNFEGYKVV LDTANGAAYT SARAVFADLE ANLTVIGENP DGLNINVKVG STHPEAMAKK VVETGSDLGL AF DGDADRL IAVDENGEIV DGDKIMFIVG KYLLGQGKLA QDTLVTTVMS NLGFHLALEE AGINSVITAV GDRYVVEEMK KNN YNFGGE QSGHMIFLDY NTTGDGQLSA IQLLKVMRET GKSLSELASE VTIYPQKLVN VRVKDNAAKK SAMDVPAIQK VISE METSM NGKGRILVRP SGTEPLLRVM AEAPTHEEVN HVVDTIVEVV EAEIGVK

UniProtKB: Phosphoglucosamine mutase

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Macromolecule #2: Diadenylate cyclase

MacromoleculeName: Diadenylate cyclase / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: diadenylate cyclase
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 32.433725 KDa
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MTDFNQFFNL EFWQKIFELN ESPLRIAIAV LDIAIVSFFL YQAIRFVQGT KLMTLVRGVI IFIFIRIIAG LIGLTTVEWL LNQVITYGV IAGVIIFQPE IRRALESLGR TTTLFTPTKK GSLDGHISAY EKSFAYMSER KIGALIAIER GQNLNEFVST G IRLDADIT ...String:
MTDFNQFFNL EFWQKIFELN ESPLRIAIAV LDIAIVSFFL YQAIRFVQGT KLMTLVRGVI IFIFIRIIAG LIGLTTVEWL LNQVITYGV IAGVIIFQPE IRRALESLGR TTTLFTPTKK GSLDGHISAY EKSFAYMSER KIGALIAIER GQNLNEFVST G IRLDADIT SELLINIFIP NTPLHDGAVI VQGNKIAVTS AYLPLTEKSG ISKQFGTRHR AAIGLSEVSD ALILVVSEET GG ISVAHNG EFFADISKEK FHDILVAILV PKVEKTVRGT GRIRKNKVEE KKNGK

UniProtKB: Diadenylate cyclase

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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

Concentration1 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMNaCLSodium Cloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Details: 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
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..
DetailsFull-length CdaA

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 5150 / Average exposure time: 9.0 sec. / Average electron dose: 53.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1908105
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio model (cryoSPARC v4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0-beta) / Number images used: 133430
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 5.0-beta)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 5.0-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: v3
RefinementSpace: REAL
Output model

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

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