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- PDB-7ujl: Bacteriophage Lambda Red-Beta N-terminal domain helical assembly ... -

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Basic information

Entry
Database: PDB / ID: 7ujl
TitleBacteriophage Lambda Red-Beta N-terminal domain helical assembly in complex with dsDNA
Components
  • Complementary DNA
  • Recombination protein bet
  • Template DNA
KeywordsRECOMBINATION/DNA / Annealase / Synaptase / SSAP / Single-strand annealing protein / DNA annealing intermediate / Recombinase / Two-component recombinase / Viral / DNA-binding / RECOMBINATION-DNA complex
Function / homologyBacteriophage lambda, Recombination protein bet / RecT family / RecT family / DNA recombination / DNA binding / DNA / DNA (> 10) / Recombination protein bet
Function and homology information
Biological speciesEscherichia virus Lambda
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNewing, T.P. / Tolun, G.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)GNT1184012 Australia
CitationJournal: Nat Commun / Year: 2022
Title: Redβ annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination.
Authors: Timothy P Newing / Jodi L Brewster / Lucy J Fitschen / James C Bouwer / Nikolas P Johnston / Haibo Yu / Gökhan Tolun /
Abstract: The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a ...The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redβ, a defining member of the Redβ/RecT protein family.
History
DepositionMar 31, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Structure summary / Category: audit_author / Item: _audit_author.name
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
A: Recombination protein bet
B: Template DNA
C: Complementary DNA


Theoretical massNumber of molelcules
Total (without water)37,8713
Polymers37,8713
Non-polymers00
Water00
1
A: Recombination protein bet
B: Template DNA
C: Complementary DNA
x 60


Theoretical massNumber of molelcules
Total (without water)2,272,241180
Polymers2,272,241180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation59
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 60 / Rise per n subunits: 2.078 Å / Rotation per n subunits: -12.947 °)

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Components

#1: Protein Recombination protein bet / Red-beta annealase


Mass: 21275.008 Da / Num. of mol.: 1 / Fragment: N-terminal domain (UNP residues 1-177)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia virus Lambda / Gene: bet, betA, red-beta, redB / Plasmid: pET24a
Details (production host): pET24a containing the truncated N-terminal 177-residue fragment of the bet gene followed by a SSHHHHHH tag under the control of the lac repressor system
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03698
#2: DNA chain Template DNA


Mass: 8244.295 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain Complementary DNA


Mass: 8351.386 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotidesCOMPLEXHelical complex assembled through sequential addition of complementary oligonucleotides in a controlled environmentall0MULTIPLE SOURCES
2Red-beta annealase N-terminal domainCOMPLEX#11RECOMBINANT
3Template ssDNACOMPLEX27mer ssDNA oligonucleotide#21RECOMBINANT
4Complementary ssDNACOMPLEX27mer ssDNA oligonucleotide#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11114.18 kDa/nmNO
21102.26 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia virus Lambda10710
33Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
22Escherichia coli BL21(DE3) (bacteria)469008pET24a
34Escherichia coli (E. coli)562
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMpotassium phosphateKH2PO41
25 mMmagnesium chlorideMgCl21
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 298 K
Details: Sample loading volume ranged between 2 and 3 microlitres. Samples were blotted for 5 seconds prior to vitrification.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 59500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4710
EM imaging opticsEnergyfilter name: GIF Quantum LS / Details: Installed but not used
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.3/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Windows / Type: package
EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
4cryoSPARC3CTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12RELION3.1classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -12.947 ° / Axial rise/subunit: 2.078 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 922280
Details: 922280 particles were initially selected using cryoSPARC filament tracing
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26525 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

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