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- EMDB-41579: Structure of full-length LexA bound to a RecA filament -

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Basic information

Entry
Database: EMDB / ID: EMD-41579
TitleStructure of full-length LexA bound to a RecA filament
Map dataSharpened map used for model building and refinement
Sample
  • Complex: SOS Signal Complex consisting of RecA, ssDNA, and LexA
    • Complex: RecA* activated filament
      • Protein or peptide: Protein RecA
      • DNA: DNA (27-MER)
    • Complex: LexA Dimer
      • Protein or peptide: LexA repressor
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsDamage 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-binding transcription factor binding / DNA recombination / 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 / cytosol / cytoplasm
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 bacterial DNA recombination protein / 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
LexA repressor / Protein RecA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsCory MB / Li A / Kohli RM
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41579.png

Downloads & links

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Map

FileDownload / File: emd_41579.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used for model building and refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 256 pix.
= 353.28 Å		1.38 Å/pix.
x 256 pix.
= 353.28 Å		1.38 Å/pix.
x 256 pix.
= 353.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0127
Minimum - Maximum-0.008536006 - 2.2161245
Average (Standard dev.)0.0019664625 (±0.030175203)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 353.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened final map from data processing

Fileemd_41579_additional_1.map
AnnotationUnsharpened final map from data processing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of final map

Fileemd_41579_half_map_1.map
AnnotationHalf map of final map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of final map

Fileemd_41579_half_map_2.map
AnnotationHalf map of final map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SOS Signal Complex consisting of RecA, ssDNA, and LexA

EntireName: SOS Signal Complex consisting of RecA, ssDNA, and LexA
Components
  • Complex: SOS Signal Complex consisting of RecA, ssDNA, and LexA
    • Complex: RecA* activated filament
      • Protein or peptide: Protein RecA
      • DNA: DNA (27-MER)
    • Complex: LexA Dimer
      • Protein or peptide: LexA repressor
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: SOS Signal Complex consisting of RecA, ssDNA, and LexA

SupramoleculeName: SOS Signal Complex consisting of RecA, ssDNA, and LexA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 480 KDa

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Supramolecule #2: RecA* activated filament

SupramoleculeName: RecA* activated filament / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12

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Supramolecule #3: LexA Dimer

SupramoleculeName: LexA Dimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12

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Macromolecule #1: Protein RecA

MacromoleculeName: Protein RecA / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 41.119551 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH HHHHHHSSGE NLYFQGMAID ENKQKALAAA LGQIEKQFGK GSIMRLGEDR SMDVETISTG SLSLDIALGA GGLPMGRIV EIYGPESSGK TTLTLQVIAA AQREGKTCAF IDAEHALDPI YARKLGVDID NLLCSQPDTG EQALEICDAL A RSGAVDVI ...String:
MGSSHHHHHH HHHHHHSSGE NLYFQGMAID ENKQKALAAA LGQIEKQFGK GSIMRLGEDR SMDVETISTG SLSLDIALGA GGLPMGRIV EIYGPESSGK TTLTLQVIAA AQREGKTCAF IDAEHALDPI YARKLGVDID NLLCSQPDTG EQALEICDAL A RSGAVDVI VVDSVAALTP KAEIEGEIGD SHMGLAARMM SQAMRKLAGN LKQSNTLLIF INQIRMKIGV MFGNPETTTG GN ALKFYAS VRLDIRRIGA VKEGENVVGS ETRVKVVKNK IAAPFKQAEF QILYGEGINF YGELVDLGVK EKLIEKAGAW YSY KGEKIG QGKANATAWL KDNPETAKEI EKKVRELLLS NPNSTPDFSV DDSEGVAETN EDF

UniProtKB: Protein RecA

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Macromolecule #2: LexA repressor

MacromoleculeName: LexA repressor / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 22.612887 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMKALTAR QQEVFDLIRD HISQTGMPPT RAEIAQRLGF RSPNAAEEHL KALARKGVIE IVSGASRGIR LLQEEEEGLP LVGRVAAGE PLLAQQHIEG HYQVDPSLFK PNADFLLRVS GMSMKDIGIM DGDLLAVHKT QDVRNGQVVV ARIDDEVTVA R LKKQGNKV ...String:
GSHMKALTAR QQEVFDLIRD HISQTGMPPT RAEIAQRLGF RSPNAAEEHL KALARKGVIE IVSGASRGIR LLQEEEEGLP LVGRVAAGE PLLAQQHIEG HYQVDPSLFK PNADFLLRVS GMSMKDIGIM DGDLLAVHKT QDVRNGQVVV ARIDDEVTVA R LKKQGNKV ELLPENSEFK PIVVDLRQQS FTIEGLAVGV IRNGDWL

UniProtKB: LexA repressor

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Macromolecule #3: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.618482 KDa
SequenceString:
(DT)(DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG)(DT) (DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG)(DT)(DG) (DG)(DT)(DG)(DG)(DT)(DG)(DG)

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 8 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
70.0 mMC4H11NO3tris
150.0 mMNaClsodium chloride
1.0 mMMgCl2magnesium chloride
2.0 mMC9H15O6PTCEP
0.25 mMC10H12Li4N5O12P3SATP gamma S tetralithium

Details: 70 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 2 mM TCEP, 0.25 mM ATPyS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: 25 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time of 7.0 s; 0.0 blot force.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsPreliminary grid screening was done manually
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2748 / Average exposure time: 5.35 sec. / Average electron dose: 46.2 e/Å2 / Details: 40 frames collected per movie
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 16.23 Å
Applied symmetry - Helical parameters - Δ&Phi: 59.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.3) / Software - details: Local Refinement / Number images used: 233920
Segment selectionNumber selected: 3423408 / Software - Name: cryoSPARC (ver. 4.0.3) / Software - details: Filament Tracer
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.
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The initial model was generated via benchling plugin for full-length construct sequence
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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