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- PDB-8trg: Structure of full-length LexA bound to a RecA filament -

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Basic information

Entry
Database: PDB / ID: 8trg
TitleStructure of full-length LexA bound to a RecA filament
Components
  • DNA (27-MER)
  • LexA repressor
  • Protein RecA
KeywordsSIGNALING PROTEIN/DNA / Damage response / signal transduction / SIGNALING PROTEIN / SIGNALING PROTEIN-DNA complex
Function / homology
Function and homology information


repressor LexA / DNA polymerase V complex / homologous recombination / SOS response / recombinational repair / DNA-binding transcription repressor activity / ATP-dependent DNA damage sensor activity / response to ionizing radiation / ATP-dependent activity, acting on DNA / translesion synthesis ...repressor LexA / DNA polymerase V complex / homologous recombination / SOS response / recombinational repair / DNA-binding transcription repressor activity / ATP-dependent DNA damage sensor activity / response to ionizing radiation / ATP-dependent activity, acting on DNA / translesion synthesis / cell motility / protein-DNA complex / single-stranded DNA binding / DNA recombination / DNA-binding transcription factor binding / sequence-specific DNA binding / damaged DNA binding / DNA replication / transcription cis-regulatory region binding / serine-type endopeptidase activity / DNA repair / negative regulation of DNA-templated transcription / DNA-templated transcription / DNA damage response / ATP hydrolysis activity / proteolysis / DNA binding / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
LexA repressor, DNA-binding domain / Transcription regulator LexA / LexA DNA binding domain / Peptidase S24, LexA-like / : / LexA-like / Peptidase S24/S26A/S26B/S26C / Peptidase S24-like / LexA/Signal peptidase-like superfamily / DNA recombination/repair protein RecA, conserved site ...LexA repressor, DNA-binding domain / Transcription regulator LexA / LexA DNA binding domain / Peptidase S24, LexA-like / : / LexA-like / Peptidase S24/S26A/S26B/S26C / Peptidase S24-like / LexA/Signal peptidase-like superfamily / DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / : / RecA C-terminal domain / recA signature. / DNA recombination and repair protein RecA / : / RecA N-terminal domain / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / LexA repressor / Protein RecA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsCory, M.B. / Li, A. / Kohli, R.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM127593 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.
Authors: Michael B Cory / Allen Li / Christina M Hurley / Peter J Carman / Ruth A Pumroy / Zachary M Hostetler / Ryann M Perez / Yarra Venkatesh / Xinning Li / Kushol Gupta / E James Petersson / Rahul M Kohli /
Abstract: The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein ...The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.
History
DepositionAug 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein RecA
B: Protein RecA
C: Protein RecA
D: Protein RecA
E: Protein RecA
F: Protein RecA
G: Protein RecA
H: Protein RecA
I: LexA repressor
J: LexA repressor
L: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)387,18127
Polymers382,80111
Non-polymers4,38016
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, assay for oligomerization, Fluorescence anisotropy experiments for complex formation
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Protein RecA


Mass: 41119.551 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: recA / Plasmid: pET41 / Production host: Escherichia coli (E. coli) / Strain (production host): BLR / Variant (production host): DE3 / References: UniProt: P0A7G6
#2: Protein LexA repressor


Mass: 22612.887 Da / Num. of mol.: 2 / Mutation: K156A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: lexA / Plasmid: pET41 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A7C2
#3: DNA chain DNA (27-MER)


Mass: 8618.482 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Synthetic oligo / Source: (synth.) Escherichia coli (E. coli)
#4: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SOS Signal Complex consisting of RecA, ssDNA, and LexACOMPLEX#1-#30RECOMBINANT
2RecA* activated filamentCOMPLEX#1, #31RECOMBINANT
3LexA DimerCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
1126 kDa/nmNO
2125 kDa/nmNO
310.48 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)562K12
32Escherichia coli (E. coli)562K12
43Escherichia coli (E. coli)562K12
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)562BL21/BLR (DE3)pET41
32Escherichia coli (E. coli)562BL21/BLR (DE3)pET41
43Escherichia coli (E. coli)562BL21/BLR (DE3)pET41
Buffer solutionpH: 7.5
Details: 70 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 2 mM TCEP, 0.25 mM ATPyS
Buffer component
IDConc.NameFormulaBuffer-ID
170 mMtrisC4H11NO31
2150 mMsodium chlorideNaCl1
31 mMmagnesium chlorideMgCl21
42 mMTCEPC9H15O6P1
50.25 mMATP gamma S tetralithiumC10H12Li4N5O12P3S1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: Blotting time of 7.0 s; 0.0 blot force

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was done manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.35 sec. / Electron dose: 46.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2748 / Details: 40 frames collected per movie

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.0.3particle selectionFilament Tracer
2EPUimage acquisition
4cryoSPARC4.0.3CTF correctionPatch CTF Estimation
7UCSF ChimeraX1.3model fitting
9PHENIX1.20.1model refinement
10ISOLDE1.4model refinement
13cryoSPARC4.0.3classification3D Classification
14cryoSPARC4.0.33D reconstructionLocal Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
1159.216.23C1
2159.216.23C1
3159.216.23C1
4159.216.23C1
Particle selectionNum. of particles selected: 3423408
Details: cryoSPARC automated Filament Tracer using filament diameter of 100A, 17A separation between segments. Minimum and maximum filament diameters of 90A, 150A respectively. Inspect Picks to ...Details: cryoSPARC automated Filament Tracer using filament diameter of 100A, 17A separation between segments. Minimum and maximum filament diameters of 90A, 150A respectively. Inspect Picks to filter particles with high curvature or sinuosity Local power > 567.940 Local power < 5711.103 Curvature (1/A) < 0.005970 Sinuosity < 1.393924 Low pass filtered particles near micrograph edge.
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233920 / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: The initial model was generated via benchling plugin for full-length construct sequence
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00523259
ELECTRON MICROSCOPYf_angle_d0.65531479
ELECTRON MICROSCOPYf_dihedral_angle_d12.7623439
ELECTRON MICROSCOPYf_chiral_restr0.0453589
ELECTRON MICROSCOPYf_plane_restr0.0063947

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