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- PDB-8zek: Cryo-EM structure of the E. coli BrxX methyltransferase complexed... -

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Basic information

Entry
Database: PDB / ID: 8zek
TitleCryo-EM structure of the E. coli BrxX methyltransferase complexed with Ocr
Components
  • Protein Ocr
  • site-specific DNA-methyltransferase (adenine-specific)
KeywordsTRANSFERASE / methyltransferase / complex
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 / 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 / : / 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
site-specific DNA-methyltransferase (adenine-specific) / Protein Ocr
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsZhu, L. / Xu, T.H. / Sun, L.T.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32271314 China
National Natural Science Foundation of China (NSFC)31600593 China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Ocr-mediated suppression of BrxX unveils a phage counter-defense mechanism.
Authors: Shen Li / Tianhao Xu / Xinru Meng / Yujuan Yan / Ying Zhou / Lei Duan / Yulong Tang / Li Zhu / Litao Sun /
Abstract: The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial ...The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichia coli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.
History
DepositionMay 6, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: site-specific DNA-methyltransferase (adenine-specific)
B: Protein Ocr
C: Protein Ocr


Theoretical massNumber of molelcules
Total (without water)165,6813
Polymers165,6813
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein site-specific DNA-methyltransferase (adenine-specific)


Mass: 138043.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: pglX, B6R15_003960 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A5E9SEK5, site-specific DNA-methyltransferase (adenine-specific)
#2: Protein Protein Ocr / Gene product 0.3 / Gp0.3 / Overcome classical restriction / Ocr


Mass: 13819.015 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T7 (virus) / Gene: 0.3 / Production host: Escherichia coli (E. coli) / References: UniProt: P03775
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: The complex of E. coli BrxX methyltransferase with Ocr from bacteriophage T7
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 3000 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 448285 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementHighest resolution: 3.15 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211809
ELECTRON MICROSCOPYf_angle_d0.61815981
ELECTRON MICROSCOPYf_dihedral_angle_d3.8241560
ELECTRON MICROSCOPYf_chiral_restr0.0381720
ELECTRON MICROSCOPYf_plane_restr0.0032089

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