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- PDB-9nv3: Hybrid model of a complex of BREX proteins BrxB and PglZ from Sal... -

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Basic information

Entry
Database: PDB / ID: 9nv3
TitleHybrid model of a complex of BREX proteins BrxB and PglZ from Salmonella typhimurium
Components
  • DUF1788 domain-containing protein
  • PglZ domain-containing protein
KeywordsANTIMICROBIAL PROTEIN / Restriction / Bacteriophage / Defense / BREX / Salmonella typhimurium / Salmonella / AlphaFold
Function / homologyAlkaline phosphatase-like protein PglZ / BREX protein BrxB / BREX protein BrxB / PglZ domain / PglZ domain / Alkaline-phosphatase-like, core domain superfamily / PglZ domain-containing protein / DUF1788 domain-containing protein
Function and homology information
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. D23580 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.45 Å
AuthorsDoyle, L.A. / Stoddard, B. / Blower, T.R. / Kaiser, B.
Funding support United Kingdom, United States, 7items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/Y003659/1 United Kingdom
The Lister Institute of Preventive Medicine United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T008695/1 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM148166 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140375 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)5 P30 CA015704-49 United States
Other private
CitationJournal: Nucleic Acids Res / Year: 2025
Title: PglZ from Type I BREX phage defence systems is a metal-dependent nuclease that forms a sub-complex with BrxB.
Authors: Jennifer J Readshaw / Lindsey A Doyle / Maria Puiu / Abigail Kelly / Andrew Nelson / Alex J Kaiser / Sydney F McGuire / Julieta Peralta Acosta / Darren L Smith / Barry L Stoddard / Brett K ...Authors: Jennifer J Readshaw / Lindsey A Doyle / Maria Puiu / Abigail Kelly / Andrew Nelson / Alex J Kaiser / Sydney F McGuire / Julieta Peralta Acosta / Darren L Smith / Barry L Stoddard / Brett K Kaiser / Tim R Blower /
Abstract: BREX (Bacteriophage Exclusion) systems, identified through shared identity with Pgl (Phage Growth Limitation) systems, are a widespread, highly diverse group of phage defence systems found throughout ...BREX (Bacteriophage Exclusion) systems, identified through shared identity with Pgl (Phage Growth Limitation) systems, are a widespread, highly diverse group of phage defence systems found throughout bacteria and archaea. The varied BREX Types harbour multiple protein subunits (between four and eight) and all encode a conserved putative phosphatase, PglZ, and an equally conserved, putative ATPase, BrxC. Almost all BREX systems also contain a site-specific methyltransferase, PglX. Despite having determined the structure and fundamental biophysical and biochemical behaviours of several BREX factors (including the PglX methyltransferase, the BrxL effector, the BrxA DNA-binding protein, and a commonly-associated transcriptional regulator, BrxR), the mechanism by which BREX impedes phage replication remains largely undetermined. In this study, we identified a stable BREX sub-complex of PglZ:BrxB, generated and validated a structural model of that protein complex, and assessed the biochemical activity of PglZ from BREX, revealing it to be a metal-dependent nuclease. PglZ can cleave cyclic oligonucleotides, linear oligonucleotides, plasmid DNA and both non-modified and modified linear phage genomes. PglZ nuclease activity has no obvious role in BREX-dependent methylation, but does contribute to BREX phage defence. BrxB binding does not impact PglZ nuclease activity. These data contribute to our growing understanding of BREX phage defence.
History
DepositionMar 20, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 2, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 9, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _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: DUF1788 domain-containing protein
C: PglZ domain-containing protein


Theoretical massNumber of molelcules
Total (without water)125,5332
Polymers125,5332
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Mass Photometry also performed
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DUF1788 domain-containing protein


Mass: 22867.256 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. D23580 (bacteria)
Gene: STMMW_44421 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6C7ILK3
#2: Protein PglZ domain-containing protein


Mass: 102665.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. D23580 (bacteria)
Gene: STMMW_44371 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6C7IK08
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: Complex of BREX proteins BrxB / PglZ from Salmonella Typhimurium str. D23580
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. D23580 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMPotassium chlorideKCl1
220 mMTris1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9417 / Details: 4686 from grid 1 and 4731 from grid 2

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionBlob picker
2cryoSPARC4.2.1particle selectionTemplate picker
3SerialEMimage acquisition
5cryoSPARC4.2.1CTF correctionPatch CTF
8UCSF ChimeraX1.9model fitting
9ISOLDE1.6model fitting
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.1classification
14cryoSPARC4.2.13D reconstruction
21PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15593169
Details: 10,876,932 from blob picking on dataset 1 4,716,237 from template picking on dataset 2
3D reconstructionResolution: 4.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123964 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: AlphaFold model, split into domains at "hinge" points, was rigid fit into the cryo-EM map. The following domains were used: (1) BrxB + 1-96 PglZ, (2) 97-292 PglZ, (3) 293-748 PglZ, and (4) ...Details: AlphaFold model, split into domains at "hinge" points, was rigid fit into the cryo-EM map. The following domains were used: (1) BrxB + 1-96 PglZ, (2) 97-292 PglZ, (3) 293-748 PglZ, and (4) 749-867 PglZ. The model was then simulated in the ISOLDE plug-in for ChimeraX to resolve distortions from rigid domain fitiing.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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