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- PDB-8emh: CryoEM characterization of a unique AAA+ BrxL phage restriction factor -
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Open data
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Basic information
Entry | Database: PDB / ID: 8emh | ||||||
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Title | CryoEM characterization of a unique AAA+ BrxL phage restriction factor | ||||||
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Function / homology | ![]() ATP-dependent peptidase activity / protein catabolic process / serine-type endopeptidase activity / ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Shen, B.W. / Stoddard, B.L. | ||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1.3 MB | Display | ![]() |
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PDB format | ![]() | 1.1 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 28248MC ![]() 8eilC ![]() 8emcC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
Experimental dataset #1 | Data reference: ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 75702.555 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: DNA chain | | Mass: 19689.639 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: DNA chain | | Mass: 19485.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: complex of walkerB mutant of BrxL with random sequence of DNA fragments Type: COMPLEX Details: 12 strands of KrxL E280Q mutant bound to short random sequence DNA fragments. Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.950 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 8 / Details: 20 mM TrisHCl, 150 mM NaCl |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Specimen support | Details: 40 / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification![]() | Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 275 K |
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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![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K |
Image recording | Average exposure time: 2 sec. / Electron dose: 30 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: DIRECT ELECTRON DE-10 (5k x 4k) / Num. of grids imaged: 2 |
Image scans | Movie frames/image: 50 / Used frames/image: 1-49 |
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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 AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 3241183 | ||||||||||||||||||||||||
Symmetry | Point symmetry![]() | ||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229430 / Num. of class averages: 4 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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