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- PDB-7ubb: Structure of RecT protein from Listeria innoccua phage A118 in co... -

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Basic information

Entry
Database: PDB / ID: 7ubb
TitleStructure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer ssDNA
ComponentsRecT
KeywordsDNA BINDING PROTEIN / DNA Recombination / DNA Annealing
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBell, C.E. / Caldwell, B.J.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: RecT
D: RecT
E: RecT
F: RecT
G: RecT
H: RecT
I: RecT
J: RecT


Theoretical massNumber of molelcules
Total (without water)247,5138
Polymers247,5138
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Native Mass Spectrometry shows two types of complexes for this sample (in ammonium acetate buffer): 7-10 subunits on on copy of the 83-mer ssDNA, or 16-17 subunits on two ...Evidence: mass spectrometry, Native Mass Spectrometry shows two types of complexes for this sample (in ammonium acetate buffer): 7-10 subunits on on copy of the 83-mer ssDNA, or 16-17 subunits on two copies of the ssDNA. It is unclear which type of complex is present in the reconstruction.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RecT


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 detailsThe 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

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RecT protein from Listeria innocua phage A118, complexed with and 83-mer ssDNA
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
Molecular weightValue: 0.602 MDa / Experimental value: NO
Source (natural)Organism: Listeria innocua Clip11262 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 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
IDConc.NameFormulaBuffer-ID
120 mMPotassium phosphateKH2PO41
210 mMMagnesium chlorideMgCl21
30.075 mMn-dodecyl-beta-maltoside1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1.5 second blot time. Ted Pella 595 filter paper.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1000 nm / Nominal defocus min: 3500 nm / C2 aperture diameter: 50 µm
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Details: Gatan BioContinuum energy filter / Energyfilter slit width: 20 eV / Spherical aberration corrector: Cs corrector was used
Image scansWidth: 6000 / Height: 4000

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC2.15.0CTF correction
7Coot0.8.7model fitting
12cryoSPARC2.15.03D reconstruction
13PHENIX1.20.1-4487model refinement
CTF correctionDetails: Patch CTD estimation was implemented in cryoSPARC with am amplitude contrast of 0.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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 buildingProtocol: 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.

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