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Yorodumi- PDB-7ubb: Structure of RecT protein from Listeria innoccua phage A118 in co... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7ubb | ||||||
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Title | Structure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer ssDNA | ||||||
Components | RecTRectangular function | ||||||
Keywords | DNA BINDING PROTEIN / DNA Recombination / DNA Annealing | ||||||
Function / homology | DNA single-strand annealing protein RecT / RecT family / RecT family / DNA metabolic process / DNA binding / Recombinase [Bacteriophage A118] Function and homology information | ||||||
Biological species | Listeria innocua Clip11262 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å | ||||||
Authors | Bell, C.E. / Caldwell, B.J. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7ubb.cif.gz | 186.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ubb.ent.gz | 141.8 KB | Display | PDB format |
PDBx/mmJSON format | 7ubb.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ub/7ubb ftp://data.pdbj.org/pub/pdb/validation_reports/ub/7ubb | HTTPS FTP |
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-Related structure data
Related structure data | 26437MC 7ub2C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 30939.100 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source 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. Source: (gene. exp.) Listeria innocua Clip11262 (bacteria) / Strain: ATCC BAA-680 / CLIP 11262 / Gene: lin0085 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI / References: UniProt: Q92FL9 Compound details | The modeled complex consists of 8 subunits of the RecT protein, which form a helical filament on ...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. | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNARectangular function Type: COMPLEX Details: The protein was purified by Nickel affinity and anion exchange chromatography. The DNA was chemically synthesized and HPLC purified. Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.602 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Listeria innocua Clip11262 (bacteria) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21 (bacteria) | ||||||||||||||||||||
Buffer solution | pH: 6 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). | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse | ||||||||||||||||||||
Specimen support | Details: 60 seconds at 20 mA using a Pelco easiGlow / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1.5 second blot time. Ted Pella 595 filter paper. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 1000 nm / Nominal defocus min: 3500 nm / C2 aperture diameter: 50 µm |
Image recording | Average exposure time: 2.83 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1619 / Details: 45 fractions, 22.80 e-/A2/s |
EM imaging optics | Energyfilter name: GIF Bioquantum / Details: Gatan BioContinuum energy filter / Energyfilter slit width: 20 eV / Spherical aberration corrector: Cs corrector was used |
Image scans | Width: 6000 / Height: 4000 |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Details: Patch CTD estimation was implemented in cryoSPARC with am amplitude contrast of 0.1 Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1000 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180965 Details: The final resolution with a tight mask was 4.5 Angstrom Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL Details: Phenix 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. |