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- PDB-7ub2: Structure of RecT protein from Listeria innoccua phage A118 in co... -

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Basic information

Entry
Database: PDB / ID: 7ub2
TitleStructure of RecT protein from Listeria innoccua phage A118 in complex with 83-mer annealed duplex
Components
  • (DNA (49-mer)) x 2
  • RecTRectangular function
KeywordsDNA BINDING PROTEIN/DNA / DNA Recombination / DNA Annealing / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homologyDNA single-strand annealing protein RecT / RecT family / RecT family / DNA metabolic process / DNA binding / DNA / DNA (> 10) / Recombinase [Bacteriophage A118]
Function and homology information
Biological speciesListeria innocua Clip11262 (bacteria)
Escherichia virus M13
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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

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Assembly

Deposited unit
E: RecT
F: RecT
G: RecT
H: RecT
I: RecT
J: RecT
K: RecT
L: RecT
M: RecT
N: RecT
Y: DNA (49-mer)
Z: DNA (49-mer)


Theoretical massNumber of molelcules
Total (without water)339,55412
Polymers339,55412
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, The components of the complex in solution were validated by native mass spectrometry.
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "I"
d_2ens_1chain "F"
d_3ens_1chain "G"
d_4ens_1chain "H"
d_5ens_1chain "E"
d_6ens_1chain "J"
d_7ens_1chain "K"
d_8ens_1chain "L"
d_9ens_1chain "M"
d_10ens_1chain "N"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALAGLNE1 - 191
d_21ens_1ALAGLNB1 - 191
d_31ens_1ALAGLNC1 - 191
d_41ens_1ALAGLND1 - 191
d_51ens_1ALAGLNA1 - 191
d_61ens_1ALAGLNF1 - 191
d_71ens_1ALAGLNG1 - 191
d_81ens_1ALAGLNH1 - 191
d_91ens_1ALAGLNI1 - 191
d_101ens_1ALAGLNJ1 - 191

NCS oper:
IDCodeMatrixVector
1given(0.259471050623, -0.674008968341, 0.691655032863), (0.805108267153, -0.244566252467, -0.540359164182), (0.533362402171, 0.69706474502, 0.479192330072)70.9717758994, 128.651182027, -116.651000834
2given(0.615552903544, -0.682645116708, 0.393815016947), (0.749733001406, 0.353227372582, -0.559580959166), (0.242888965458, 0.639707798837, 0.729231707049)72.6934191064, 60.8606594945, -96.3233640437
3given(0.89455871663, -0.426658572495, 0.133143400205), (0.445128287168, 0.823591935978, -0.351506942967), (0.0403176197707, 0.373709493468, 0.926669144856)44.8697598141, 11.6316162461, -53.0881035145
4given(-0.0371204889288, -0.403741855299, 0.914119567443), (0.59039614233, -0.746895125321, -0.305908592383), (0.806259551524, 0.52833718973, 0.266092746096)37.1706212372, 190.451669009, -109.802830713
5given(0.893018765408, 0.447951203254, 0.0430952913052), (-0.42940885893, 0.819553729525, 0.379393880149), (0.134631038406, -0.357311354319, 0.924231074771)-43.5569253898, 29.6288821697, 47.9944244944
6given(0.612938963924, 0.750255322346, 0.247836191457), (-0.682500210803, 0.344681342205, 0.644506194377), (0.398119691498, -0.564191211938, 0.723318040432)-67.2871784214, 90.4111703146, 76.172550916
7given(0.259750899447, 0.801656015234, 0.538402362064), (-0.668707193833, -0.25291165302, 0.699189805906), (0.696677945074, -0.541648713657, 0.470379115014)-60.0415766192, 160.829547107, 76.6381819331
8given(-0.0366965995054, 0.580890424184, 0.813154151853), (-0.393086757834, -0.756498733104, 0.52267816831), (0.918768828596, -0.300459617758, 0.256101264534)-22.3268434051, 215.880475125, 53.0832677401
9given(-0.173645233714, 0.175570856924, 0.969031582048), (0.0355922946478, -0.982218252019, 0.184337988385), (0.984164885256, 0.0664994706667, 0.164308548259)34.8705320284, 236.299143425, 17.0800541798

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Components

#1: Protein
RecT / Rectangular function


Mass: 30939.100 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria innocua Clip11262 (bacteria) / Strain: ATCC BAA-680 / CLIP 11262 / Gene: lin0085 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21(AI) / References: UniProt: Q92FL9
#2: DNA chain DNA (49-mer)


Mass: 15302.170 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia virus M13
#3: DNA chain DNA (49-mer)


Mass: 14860.490 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia virus M13
Sequence detailsThe sample sequence for chain Y was modeled as dA49 because the actual sequence could not be fit to ...The sample sequence for chain Y was modeled as dA49 because the actual sequence could not be fit to the density, and only the central portion of it was modeled. The actual sample sequence is a naturally occurring 83-mer sequence from M13 DNA (5'-TTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAATTGCTGAATCTGGTGCTGTAGCTCAACATGTTTTAAATATGCAA-3'). The sequence for chain Z was modeled as dT49 because the density was not clear enough to read the sequence, and only the central portion was modeled. The true sample sequence is an 83-mer naturally occurring sequence from M13 DNA (5'-TTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAATTGCTGAATCTGGTGCTGTAGCTCAACATGTTTTAAATATGCAA-3')

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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 two complementary strands of ssDNA that were added to the protein sequentiallyRectangular 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: MULTIPLE SOURCES
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 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).
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: 20 mA with 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 2.7 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2038 / Details: 36 fractions, 24.28 e-/A2/s
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Spherical aberration corrector: Cs corrector was used
Image scansWidth: 6000 / Height: 4000

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 390000
Details: Tight Mask FSC resolution was 3.4; No mask FSC resolution was 4.3
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Phenix Real Space Refinement included secondary restraints and 10-fold NCS constraints.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.3 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002418152
ELECTRON MICROSCOPYf_angle_d0.505624901
ELECTRON MICROSCOPYf_chiral_restr0.03932722
ELECTRON MICROSCOPYf_plane_restr0.00222758
ELECTRON MICROSCOPYf_dihedral_angle_d19.62593054
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EELECTRON MICROSCOPYNCS constraints9.02942571261E-11
ens_1d_3EELECTRON MICROSCOPYNCS constraints1.6368129568E-11
ens_1d_4EELECTRON MICROSCOPYNCS constraints4.32084566718E-13
ens_1d_5EELECTRON MICROSCOPYNCS constraints2.35537735419E-13
ens_1d_6EELECTRON MICROSCOPYNCS constraints2.42014494917E-10
ens_1d_7EELECTRON MICROSCOPYNCS constraints2.36488793504E-10
ens_1d_8EELECTRON MICROSCOPYNCS constraints2.27353138599E-13
ens_1d_9EELECTRON MICROSCOPYNCS constraints3.87187036224E-13
ens_1d_10EELECTRON MICROSCOPYNCS constraints4.81513537199E-11

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