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Yorodumi- PDB-8emc: CryoEM characterization of BrxL -- a unique AAA+ phage restrictio... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8emc | ||||||
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Title | CryoEM characterization of BrxL -- a unique AAA+ phage restriction Factor. | ||||||
Components | Protease Lon-related BREX system protein BrxL | ||||||
Keywords | ANTIMICROBIAL PROTEIN / phage restriction factor / AAA+ protein. BrxL | ||||||
Function / homology | Function and homology information ATP-dependent peptidase activity / protein catabolic process / serine-type endopeptidase activity / proteolysis / ATP binding Similarity search - Function | ||||||
Biological species | Acinetobacter sp. NEB 394 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å | ||||||
Authors | Shen, B.W. / Stoddard, B.L. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nucleic Acids Res / Year: 2023 Title: Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor. Authors: Betty W Shen / Lindsey A Doyle / Rachel Werther / Abigail A Westburg / Daniel P Bies / Stephanie I Walter / Yvette A Luyten / Richard D Morgan / Barry L Stoddard / Brett K Kaiser / Abstract: Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been ...Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8emc.cif.gz | 1.5 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8emc.ent.gz | 1.3 MB | Display | PDB format |
PDBx/mmJSON format | 8emc.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8emc_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8emc_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8emc_validation.xml.gz | 240.9 KB | Display | |
Data in CIF | 8emc_validation.cif.gz | 362.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/em/8emc ftp://data.pdbj.org/pub/pdb/validation_reports/em/8emc | HTTPS FTP |
-Related structure data
Related structure data | 28244MC 8eilC 8emhC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 75703.539 Da / Num. of mol.: 14 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acinetobacter sp. NEB 394 (bacteria) / Gene: brxL, HUK62_18280 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7H8SL14 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: dimer of heptamer complex of BrxL / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 1.05 MDa / Experimental value: NO |
Source (natural) | Organism: Acinetobacter (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 / Details: 20 mM TrisHCl, 150 mM NaCl |
Specimen | Conc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-dispersed |
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 38000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: DIRECT ELECTRON DE-10 (5k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3000 Details: 2980 movies at 1.12 pixel was extracted at box size of 406 pix, fourier cropped to box size of 392 pix and combined with 100 movies collected at 1.16 pix size |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 96079 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C7 (7 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89373 / Num. of class averages: 93 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||
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