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- PDB-9ewz: Cryo-EM structure of the E. coli BrxX methyltransferase in comple... -

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Basic information

Entry
Database: PDB / ID: 9ewz
TitleCryo-EM structure of the E. coli BrxX methyltransferase in complex with DNA
Components
  • (DNA (26-MER) with BREX binding site GGTAAG) x 2
  • Adenine-specific methyltransferase BrxX
KeywordsTRANSFERASE / Methyltransferase / phage defense / BREX
Function / homology
Function and homology information


DNA modification / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / methylation / defense response to virus / nucleic acid binding
Similarity search - Function
: / Type II restriction enzyme and methyltransferase RM.Eco57I-like / Eco57I restriction-modification methylase / : / PTS HPR domain serine phosphorylation site signature. / N-6 Adenine-specific DNA methylases signature. / DNA methylase, N-6 adenine-specific, conserved site / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / Adenine-specific methyltransferase BrxX
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.22 Å
AuthorsAdams, M.C. / Ghilarov, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust221868/Z/20/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01/097X/1 United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Molecular basis of foreign DNA recognition by BREX anti-phage immunity system.
Authors: Alena Drobiazko / Myfanwy C Adams / Mikhail Skutel / Kristina Potekhina / Oksana Kotovskaya / Anna Trofimova / Mikhail Matlashov / Daria Yatselenko / Karen L Maxwell / Tim R Blower / ...Authors: Alena Drobiazko / Myfanwy C Adams / Mikhail Skutel / Kristina Potekhina / Oksana Kotovskaya / Anna Trofimova / Mikhail Matlashov / Daria Yatselenko / Karen L Maxwell / Tim R Blower / Konstantin Severinov / Dmitry Ghilarov / Artem Isaev /
Abstract: Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on site-specific epigenetic DNA methylation to discriminate between the host and invading DNA. We demonstrate that in Type I ...Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on site-specific epigenetic DNA methylation to discriminate between the host and invading DNA. We demonstrate that in Type I BREX systems, defense and methylation require BREX site DNA binding by the BrxX (PglX) methyltransferase employing S-adenosyl methionine as a cofactor. We determined 2.2-Å cryoEM structure of Escherichia coli BrxX bound to target dsDNA revealing molecular details of BREX DNA recognition. Structure-guided engineering of BrxX expands its DNA specificity and dramatically enhances phage defense. We show that BrxX alone does not methylate DNA, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters BrxX in an inactive dimeric form. We propose that BrxX-mediated foreign DNA sensing is a necessary first step in activation of BREX defense.
History
DepositionApr 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Adenine-specific methyltransferase BrxX
B: DNA (26-MER) with BREX binding site GGTAAG
A: DNA (26-MER) with BREX binding site GGTAAG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,9085
Polymers153,4853
Non-polymers4232
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 Adenine-specific methyltransferase BrxX / BREX protein PglX


Mass: 138128.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: pglX, brxX, EcHS_A0339 / Production host: Escherichia coli B (bacteria)
References: UniProt: P0DUF9, site-specific DNA-methyltransferase (adenine-specific)
#2: DNA chain DNA (26-MER) with BREX binding site GGTAAG


Mass: 7803.045 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (26-MER) with BREX binding site GGTAAG


Mass: 7553.886 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1BrxX methyltransferase from E. coli BREX phage defense system in complex with DNACOMPLEX#1-#30MULTIPLE SOURCES
2BrxX methyltransferaseCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli B (bacteria)37762
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 50.5 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 216233 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211119
ELECTRON MICROSCOPYf_angle_d0.46815252
ELECTRON MICROSCOPYf_dihedral_angle_d15.4484208
ELECTRON MICROSCOPYf_chiral_restr0.0381649
ELECTRON MICROSCOPYf_plane_restr0.0031801

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