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- PDB-8emc: CryoEM characterization of BrxL -- a unique AAA+ phage restrictio... -

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Basic information

Entry
Database: PDB / ID: 8emc
TitleCryoEM characterization of BrxL -- a unique AAA+ phage restriction Factor.
ComponentsProtease Lon-related BREX system protein BrxL
KeywordsANTIMICROBIAL 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
Lon-like protease BrxL / Lon-like protease BrxL-like / BREX system Lon protease-like BrxL, N-terminal / Lon-like protease BrxL-like, ATPase domain / BREX system Lon protease-like protein BrxL N-terminal / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protease Lon-related BREX system protein BrxL
Similarity search - Component
Biological speciesAcinetobacter sp. NEB 394 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsShen, B.W. / Stoddard, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM105691 United States
CitationJournal: 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
DepositionSep 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 17, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protease Lon-related BREX system protein BrxL
B: Protease Lon-related BREX system protein BrxL
C: Protease Lon-related BREX system protein BrxL
D: Protease Lon-related BREX system protein BrxL
E: Protease Lon-related BREX system protein BrxL
F: Protease Lon-related BREX system protein BrxL
G: Protease Lon-related BREX system protein BrxL
H: Protease Lon-related BREX system protein BrxL
I: Protease Lon-related BREX system protein BrxL
J: Protease Lon-related BREX system protein BrxL
K: Protease Lon-related BREX system protein BrxL
L: Protease Lon-related BREX system protein BrxL
M: Protease Lon-related BREX system protein BrxL
N: Protease Lon-related BREX system protein BrxL


Theoretical massNumber of molelcules
Total (without water)1,059,85014
Polymers1,059,85014
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Protease Lon-related BREX system protein BrxL


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

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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: dimer of heptamer complex of BrxL / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.05 MDa / Experimental value: NO
Source (natural)Organism: Acinetobacter (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20 mM TrisHCl, 150 mM NaCl
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-dispersed
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7UCSF Chimeramodel fitting
9Coot9.03model refinement
10cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 96079
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89373 / Num. of class averages: 93 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00474852
ELECTRON MICROSCOPYf_angle_d0.644101000
ELECTRON MICROSCOPYf_dihedral_angle_d4.4610097
ELECTRON MICROSCOPYf_chiral_restr0.04311072
ELECTRON MICROSCOPYf_plane_restr0.00513037

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