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- PDB-8eil: C-Terminal Domain of BrxL from Acinetobacter BREX type I phage re... -

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Basic information

Entry
Database: PDB / ID: 8eil
TitleC-Terminal Domain of BrxL from Acinetobacter BREX type I phage restriction system
ComponentsProtease Lon-related BREX system protein BrxL
KeywordsDNA BINDING PROTEIN / helicase / phage restriction / LonP/RadA homolog / BREX
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
MALONIC ACID / SUCCINIC ACID / Protease Lon-related BREX system protein BrxL
Similarity search - Component
Biological speciesAcinetobacter sp. NEB 394 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.25 Å
AuthorsDoyle, L.A. / Stoddard, B.L. / Kaiser, B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R15GM140375-01A1 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 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release
Revision 1.1May 17, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)78,9407
Polymers78,6144
Non-polymers3263
Water2,162120
1
A: Protease Lon-related BREX system protein BrxL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,7582
Polymers19,6541
Non-polymers1041
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Protease Lon-related BREX system protein BrxL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,7582
Polymers19,6541
Non-polymers1041
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Protease Lon-related BREX system protein BrxL


Theoretical massNumber of molelcules
Total (without water)19,6541
Polymers19,6541
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Protease Lon-related BREX system protein BrxL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,7722
Polymers19,6541
Non-polymers1181
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)213.344, 213.344, 213.344
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number196
Space group name H-MF23
Components on special symmetry positions
IDModelComponents
11A-820-

HOH

21B-818-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid -1 through 0 or (resid 1...
21(chain B and (resid -1 through 11 or (resid 12...
31(chain C and ((resid -1 and (name N or name...
41(chain D and ((resid -1 and (name N or name...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid -1 through 0 or (resid 1...A-1 - 0
121(chain A and (resid -1 through 0 or (resid 1...A1
131(chain A and (resid -1 through 0 or (resid 1...A-1 - 182
141(chain A and (resid -1 through 0 or (resid 1...A-1 - 182
151(chain A and (resid -1 through 0 or (resid 1...A-1 - 182
161(chain A and (resid -1 through 0 or (resid 1...A-1 - 182
211(chain B and (resid -1 through 11 or (resid 12...B-1 - 11
221(chain B and (resid -1 through 11 or (resid 12...B12 - 13
231(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
241(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
251(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
261(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
271(chain B and (resid -1 through 11 or (resid 12...B15
281(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
291(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
2101(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
2111(chain B and (resid -1 through 11 or (resid 12...B-1 - 181
311(chain C and ((resid -1 and (name N or name...C-1
321(chain C and ((resid -1 and (name N or name...C-2 - 180
331(chain C and ((resid -1 and (name N or name...C-2 - 180
341(chain C and ((resid -1 and (name N or name...C-2 - 180
351(chain C and ((resid -1 and (name N or name...C-2 - 180
411(chain D and ((resid -1 and (name N or name...D-1
421(chain D and ((resid -1 and (name N or name...D-2 - 180
431(chain D and ((resid -1 and (name N or name...D-2 - 180
441(chain D and ((resid -1 and (name N or name...D-2 - 180
451(chain D and ((resid -1 and (name N or name...D-2 - 180

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Components

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


Mass: 19653.572 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter sp. NEB 394 (bacteria) / Gene: brxL / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3R9EDI8
#2: Chemical ChemComp-MLA / MALONIC ACID / DICARBOXYLIC ACID C3 / PROPANEDIOLIC ACID / METHANEDICARBOXYLIC ACID


Mass: 104.061 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H4O4
#3: Chemical ChemComp-SIN / SUCCINIC ACID


Mass: 118.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6O4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 120 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.57 Å3/Da / Density % sol: 52.2 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / Details: 45% Tacsimate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.9774 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Jul 2, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9774 Å / Relative weight: 1
ReflectionResolution: 2.25→50 Å / Num. obs: 37994 / % possible obs: 100 % / Redundancy: 39.8 % / Rmerge(I) obs: 0.159 / Rpim(I) all: 0.025 / Rrim(I) all: 0.161 / Χ2: 0.536 / Net I/σ(I): 2.9 / Num. measured all: 1513126
Reflection shell

Diffraction-ID: 1 / % possible all: 100

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2
2.25-2.2931.32.63618780.5810.4762.6790.399
2.29-2.3337.12.02718540.7640.3372.0540.422
2.33-2.3837.92.26118900.8020.3722.2910.403
2.38-2.4240.51.63119040.8670.2591.6510.406
2.42-2.4841.71.38518960.8840.2171.4020.414
2.48-2.5341.41.21418500.9090.1911.2280.416
2.53-2.640.71.00719140.9350.161.020.42
2.6-2.6739.90.8218790.9570.1310.830.428
2.67-2.75390.7219050.9720.1170.730.425
2.75-2.8341.40.56718810.980.0890.5740.444
2.83-2.9439.50.4218960.9870.0680.4260.459
2.94-3.0541.70.33918960.9910.0530.3430.482
3.05-3.19420.24718970.9950.0390.250.525
3.19-3.3640.90.17918940.9980.0280.1810.58
3.36-3.5739.20.14219160.9980.0230.1440.619
3.57-3.8540.40.1119000.9990.0180.1110.691
3.85-4.2341.50.0919020.9990.0140.0910.778
4.23-4.85410.07419240.9990.0120.0750.816
4.85-6.139.40.07219320.9990.0120.0730.716
6.1-5039.90.059198610.010.060.799

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.5 Å48.94 Å
Translation2.5 Å48.94 Å

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Processing

Software
NameVersionClassification
HKL-2000v722data scaling
PHASER2.8.3phasing
PHENIX1.18_3855refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Alphafold2 model

Resolution: 2.25→48.94 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 0 / Phase error: 27.49 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2303 1879 5.34 %
Rwork0.2162 33295 -
obs0.217 35174 92.58 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 124.64 Å2 / Biso mean: 61.6607 Å2 / Biso min: 20.73 Å2
Refinement stepCycle: final / Resolution: 2.25→48.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5066 0 22 120 5208
Biso mean--66.37 49.38 -
Num. residues----716
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2014X-RAY DIFFRACTION5.544TORSIONAL
12B2014X-RAY DIFFRACTION5.544TORSIONAL
13C2014X-RAY DIFFRACTION5.544TORSIONAL
14D2014X-RAY DIFFRACTION5.544TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.25-2.310.33991200.29872017213774
2.31-2.380.32791340.2912192232680
2.38-2.460.3561280.27792374250287
2.46-2.550.28811400.26942471261190
2.55-2.650.33371420.26462556269892
2.65-2.770.32021480.26822570271893
2.77-2.910.27331400.25272610275095
2.92-3.10.3061480.25152671281997
3.1-3.340.29451470.23682731287898
3.34-3.670.23091560.20582726288298
3.67-4.20.2041580.188727602918100
4.2-5.290.15751580.177227612919100
5.29-48.940.19491600.19612856301699

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