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- EMDB-26434: Structure of RecT protein from Listeria innoccua phage A118 in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-26434
TitleStructure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer annealed duplex
Map dataDensity modified map from Phenix-Resolve, including 10-fold NCS averaging.
Sample
  • Complex: RecT protein from Listeria innocua phage A118, complexed with two complementary strands of ssDNA that were added to the protein sequentiallyRectangular function
    • Protein or peptide: RecTRectangular function
    • DNA: DNA (49-mer)
    • DNA: DNA (49-mer)
Function / homologyDNA single-strand annealing protein RecT / RecT family / RecT family / DNA metabolic process / DNA binding / Putative recombinase [Bacteriophage A118]
Function and homology information
Biological speciesListeria innocua Clip11262 (bacteria) / Escherichia virus M13
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26434.png

Downloads & links

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Map

FileDownload / File: emd_26434.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map from Phenix-Resolve, including 10-fold NCS averaging.
Voxel sizeX=Y=Z: 0.899 Å
Density
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-1.6565504 - 2.563766
Average (Standard dev.)-5.575307e-15 (±0.065611586)
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: Un-masked half map

Fileemd_26434_half_map_1.map
AnnotationUn-masked half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Un-masked half map

Fileemd_26434_half_map_2.map
AnnotationUn-masked half 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 : RecT protein from Listeria innocua phage A118, complexed with two...

EntireName: RecT protein from Listeria innocua phage A118, complexed with two complementary strands of ssDNA that were added to the protein sequentiallyRectangular function
Components
  • Complex: RecT protein from Listeria innocua phage A118, complexed with two complementary strands of ssDNA that were added to the protein sequentiallyRectangular function
    • Protein or peptide: RecTRectangular function
    • DNA: DNA (49-mer)
    • DNA: DNA (49-mer)

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

SupramoleculeName: RecT protein from Listeria innocua phage A118, complexed with two complementary strands of ssDNA that were added to the protein sequentially
type: complex / ID: 1 / Chimera: Yes / 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 / Number of copies: 10 / 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

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Macromolecule #2: DNA (49-mer)

MacromoleculeName: DNA (49-mer) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia virus M13
Molecular weightTheoretical: 15.30217 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)

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Macromolecule #3: DNA (49-mer)

MacromoleculeName: DNA (49-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia virus M13
Molecular weightTheoretical: 14.86049 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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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 two oligonucleotides added sequentially, and placed on ice for 90 min. Then immediately prior to vitrification, 1 ul of 1.5 mM n-dodecyl-beta- ...Details: The LiRecT protein was mixed at 37C with two oligonucleotides added sequentially, 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: 20 mA with 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: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsSpherical aberration corrector: Cs corrector was used / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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: 2038 / Average exposure time: 2.7 sec. / Average electron dose: 66.0 e/Å2 / Details: 36 fractions, 24.28 e-/A2/s
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0)
Details: Tight Mask FSC resolution was 3.4; No mask FSC resolution was 4.3
Number images used: 390000
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsPhenix Real Space Refinement included secondary restraints and 10-fold NCS constraints.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ub2:
Structure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer annealed duplex

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