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- EMDB-50032: Cryo-EM structure of the E. coli BrxX methyltransferase in comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-50032
TitleCryo-EM structure of the E. coli BrxX methyltransferase in complex with Ocr
Map data
Sample
  • Complex: BrxX methyltransferase from E. coli BREX phage defense system in complex with Ocr
    • Complex: BrxX methyltransferase
      • Protein or peptide: Adenine-specific methyltransferase BrxX
    • Complex: Ocr
      • Protein or peptide: Protein Ocr
  • Ligand: MAGNESIUM ION
  • Ligand: S-ADENOSYLMETHIONINE
KeywordsMethyltransferase / phage defense / BREX / DNA-binding / TRANSFERASE
Function / homology
Function and homology information


symbiont-mediated evasion of host restriction-modification system / DNA modification / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / methylation / defense response to virus / nucleic acid binding / symbiont-mediated suppression of host innate immune response
Similarity search - Function
: / B-form DNA mimic Ocr / DNA mimic ocr / Protein Ocr / 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
Protein Ocr / Adenine-specific methyltransferase BrxX
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsAdams MC / Ghilarov D
Funding support United Kingdom, 2 items
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, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50032.png

Downloads & links

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Map

FileDownload / File: emd_50032.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 480 pix.
= 326.4 Å		0.68 Å/pix.
x 480 pix.
= 326.4 Å		0.68 Å/pix.
x 480 pix.
= 326.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0226
Minimum - Maximum-0.05402861 - 0.17152205
Average (Standard dev.)0.0006952604 (±0.004182251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 326.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : BrxX methyltransferase from E. coli BREX phage defense system in ...

EntireName: BrxX methyltransferase from E. coli BREX phage defense system in complex with Ocr
Components
  • Complex: BrxX methyltransferase from E. coli BREX phage defense system in complex with Ocr
    • Complex: BrxX methyltransferase
      • Protein or peptide: Adenine-specific methyltransferase BrxX
    • Complex: Ocr
      • Protein or peptide: Protein Ocr
  • Ligand: MAGNESIUM ION
  • Ligand: S-ADENOSYLMETHIONINE

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Supramolecule #1: BrxX methyltransferase from E. coli BREX phage defense system in ...

SupramoleculeName: BrxX methyltransferase from E. coli BREX phage defense system in complex with Ocr
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: BrxX methyltransferase

SupramoleculeName: BrxX methyltransferase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: Ocr

SupramoleculeName: Ocr / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia phage T7 (virus)

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Macromolecule #1: Adenine-specific methyltransferase BrxX

MacromoleculeName: Adenine-specific methyltransferase BrxX / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: site-specific DNA-methyltransferase (adenine-specific)
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 138.128328 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString: MNTNNIKKYA PQARNDFRDA VIQKLTTLGI AADKKGNLQI AEAETIGETV RYGQFDYPLS TLPRRERLVK RAREQGFEVL VEHCAYTWF NRLCAIRYME LHGYLDHGFR MLSHPETPTA FEVLDHVPEV AEALLPESKA QLVEMKLSGN QDEALYRELL L GQCHALHH ...String:
MNTNNIKKYA PQARNDFRDA VIQKLTTLGI AADKKGNLQI AEAETIGETV RYGQFDYPLS TLPRRERLVK RAREQGFEVL VEHCAYTWF NRLCAIRYME LHGYLDHGFR MLSHPETPTA FEVLDHVPEV AEALLPESKA QLVEMKLSGN QDEALYRELL L GQCHALHH AMPFLFEAVD DEAELLLPDN LTRTDSILRG LVDDIPEEDW EQVEVIGWLY QFYISEKKDA VIGKVVKSED IP AATQLFT PNWIVQYLVQ NSVGRQWLQT YPDSPLKDKM EYYIEPAEQT PEVQAQLAAI TPASIEPESI KVLDPACGSG HIL TEAYNV LKAIYEERGY RTRDIPQLIL ENNIFGLDID DRAAQLSGFA MLMLARQDDR RILGRGVRLN IVSLQESKLD IAEV WTKLN FHQHMQRGSM GDMFTQGTAL ANTDSAEYKL LMRTLALFTS AKTLGSLIQV PQEDEAALKA FLERLYRLAV EGDIQ QKEA AAELIPYIQQ AWILAQRYDA VVANPPYMGG KGMNGDLKEF AKKQFPDSKS DLFAMFMQHA FSLLKENGFN AQVNMQ SWM FLSSYEALRG WLLDNKTFIT MAHLGARAFG QISGEVVQTT AWVIKNNHSG FYKPVFFRLV DDNEEHKKNN LLNRMNC FK NTLQNDFKKI PGSPIAYWAT LAFINSFLKL PALGTRAVKG LDTNGSIDVF LRRWPEVSIN SFDALGKGNS KWFPIAKG G ELRKWFGNHE YIINYENDGI ELRKNKANLR NKDMYFQEGG TWTVVSTTGF SMRYMPKGFL FDQGGSAVFC ENNDELSIY NILACMNSKY INYSASLICP TLNFTTGDVR KFPVIKNNHL EDLAKKAIEI SKADWNQFET SWEFSKNKLI EHKGNVAYSY ASYCNFQDK LYEQLVNIEK NINNIIEEIL GFKIETTENS ELITLNSNKI YRYGQSETND TFLNRHRSDT ISELISYSVG C QMGRYSLD REGLVYAHEG NKGFAELAAE GAYKTFPADN DGILPLMDDE WFEDDVTSRV KEFVRTVWGE EHLQENLEFI AE SLCLYAI KPKKGESALE TIRRYLSTQF WKDHMKMYKK RPIYWLFSSG KEKAFECLVY LHRYNDATLS RMRTEYVVPL LAR YQANID RLNDQLDEAS GGEATRLKRE RDSLIKKFSE LRSYDDRLRH YADMRISIDL DDGVKVNYGK FGDLLADVKA ITGN APEAI

UniProtKB: Adenine-specific methyltransferase BrxX

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Macromolecule #2: Protein Ocr

MacromoleculeName: Protein Ocr / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage T7 (virus)
Molecular weightTheoretical: 13.819015 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString:
MAMSNMTYNN VFDHAYEMLK ENIRYDDIRD TDDLHDAIHM AADNAVPHYY ADIFSVMASE GIDLEFEDSG LMPDTKDVIR ILQARIYEQ LTIDLWEDAE DLLNEYLEEV EEYEEDEE

UniProtKB: Protein Ocr

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: S-ADENOSYLMETHIONINE

MacromoleculeName: S-ADENOSYLMETHIONINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: SAM
Molecular weightTheoretical: 398.437 Da
Chemical component information

ChemComp-SAM:
S-ADENOSYLMETHIONINE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 35.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 94764
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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