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- EMDB-26437: Structure of RecT protein from Listeria innoccua phage A118 in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-26437
TitleStructure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer ssDNA
Map dataDensity Modified Map from PHENIX RESOLVE procedure
Sample
  • Complex: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNARectangular function
    • Protein or peptide: RecTRectangular function
KeywordsDNA Recombination / DNA Annealing / DNA BINDING PROTEIN
Function / homologyDNA single-strand annealing protein RecT / RecT family / RecT family / DNA metabolic process / DNA binding / Recombinase [Bacteriophage A118]
Function and homology information
Biological speciesListeria innocua Clip11262 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBell CE / Caldwell BJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1616105 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing.
Authors: Brian J Caldwell / Andrew S Norris / Caroline F Karbowski / Alyssa M Wiegand / Vicki H Wysocki / Charles E Bell /
Abstract: Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, ...Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redβ from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redβ homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.
History
DepositionMar 14, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26437.png

Downloads & links

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Map

FileDownload / File: emd_26437.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity Modified Map from PHENIX RESOLVE procedure
Voxel sizeX=Y=Z: 0.899 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-1.0352142 - 1.412036
Average (Standard dev.)0.000000000000168 (±0.0256524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 251.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Unmasked half-map from cryoSPARC

Fileemd_26437_half_map_1.map
AnnotationUnmasked half-map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked half-map from cryoSPARC

Fileemd_26437_half_map_2.map
AnnotationUnmasked half-map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RecT protein from Listeria innocua phage A118, complexed with and...

EntireName: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNARectangular function
Components
  • Complex: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNARectangular function
    • Protein or peptide: RecTRectangular function

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Supramolecule #1: RecT protein from Listeria innocua phage A118, complexed with and...

SupramoleculeName: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The protein was purified by Nickel affinity and anion exchange chromatography. The DNA was chemically synthesized and HPLC purified.
Source (natural)Organism: Listeria innocua Clip11262 (bacteria)
Molecular weightTheoretical: 602 KDa

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Macromolecule #1: RecT

MacromoleculeName: RecT / type: protein_or_peptide / ID: 1
Details: The modeled complex consists of 8 subunits of the RecT protein, which form a helical filament on ssDNA. The ssDNA was not modeled due to the low resolution. Density for four additional ...Details: The modeled complex consists of 8 subunits of the RecT protein, which form a helical filament on ssDNA. The ssDNA was not modeled due to the low resolution. Density for four additional subunits, two on either end of the filament, was present, but not clear enough to dock into.
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Listeria innocua Clip11262 (bacteria) / Strain: ATCC BAA-680 / CLIP 11262
Molecular weightTheoretical: 30.9391 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSHMATNDEL KNQLANKQNG GQVASAQSLD LKGLLEAPTM RKKFEKVLDK KAPQFLTSLL NLYNGDDYLQ KTDPMTVVTS AMVAATLDL PIDKNLGYAW IVPYKGRAQF QLGYKGYIQL ALRTGQYKSI NVIEVREGEL LKWNRLTEEI ELDLDNNTSE K VVGYCGYF ...String:
GSHMATNDEL KNQLANKQNG GQVASAQSLD LKGLLEAPTM RKKFEKVLDK KAPQFLTSLL NLYNGDDYLQ KTDPMTVVTS AMVAATLDL PIDKNLGYAW IVPYKGRAQF QLGYKGYIQL ALRTGQYKSI NVIEVREGEL LKWNRLTEEI ELDLDNNTSE K VVGYCGYF QLINGFEKTV YWTRKEIEAH KQKFSKSDFG WKKDYDAMAK KTVLRNMLSK WGILSIDMQT AVTEDEAEPR ER KDVTDDE SIPDIIDAPV TPSDTLEAGS VVQGSMI

UniProtKB: Recombinase [Bacteriophage A118]

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
20.0 mMKH2PO4Potassium phosphate
10.0 mMMgCl2Magnesium chloride
0.075 mMn-dodecyl-beta-maltoside

Details: The LiRecT protein was mixed at 37C with one oligonucleotide, and placed on ice for 90 min. Then immediately prior to vitrification, 1 ul of 1.5 mM n-dodecyl-beta-maltoside (Anatrace) was added (0.5 CMC).
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 60 seconds at 20 mA using a Pelco easiGlow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 1.5 second blot time. Ted Pella 595 filter paper..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 81000
Specialist opticsSpherical aberration corrector: Cs corrector was used / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV / Details: Gatan BioContinuum energy filter
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 1619 / Average exposure time: 2.83 sec. / Average electron dose: 65.0 e/Å2 / Details: 45 fractions, 22.80 e-/A2/s
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1000
Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0)
Details: The final resolution with a tight mask was 4.5 Angstrom
Number images used: 180965
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsPhenix Real Space Refinement was by rigid body refinement of the eight different subunits. Due to the limited resolution, the refinement was limited to rigid body only.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ubb:
Structure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer ssDNA

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