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- PDB-8u0o: Synaptic complex of human DNA polymerase Lambda DL variant engage... -

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Basic information

Entry
Database: PDB / ID: 8u0o
TitleSynaptic complex of human DNA polymerase Lambda DL variant engaged on a DNA double-strand break containing an unpaired 3' primer terminus
Components
  • DNA (5'-D(*AP*CP*GP*CP*GP*GP*CP*A)-3')
  • DNA (5'-D(P*GP*CP*CP*GP*CP*GP*TP*A)-3')
  • DNA polymerase lambda
KeywordsTRANSFERASE/DNA / NONHOMOLOGOUS END-JOINING / BASE EXCISION REPAIR / DNA POLYMERASE / TRANSFERASE / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA biosynthetic process / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / base-excision repair, gap-filling / nucleotide-excision repair / Nonhomologous End-Joining (NHEJ) / double-strand break repair via homologous recombination / double-strand break repair via nonhomologous end joining / site of double-strand break ...DNA biosynthetic process / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / base-excision repair, gap-filling / nucleotide-excision repair / Nonhomologous End-Joining (NHEJ) / double-strand break repair via homologous recombination / double-strand break repair via nonhomologous end joining / site of double-strand break / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
DNA polymerase family X, beta-like / DNA polymerase beta, palm domain / DNA polymerase beta palm / DNA polymerase lambda, fingers domain / Fingers domain of DNA polymerase lambda / DNA-directed DNA polymerase X / DNA polymerase beta-like, N-terminal domain / Helix-hairpin-helix domain / DNA polymerase X family / DNA polymerase lambda lyase domain superfamily ...DNA polymerase family X, beta-like / DNA polymerase beta, palm domain / DNA polymerase beta palm / DNA polymerase lambda, fingers domain / Fingers domain of DNA polymerase lambda / DNA-directed DNA polymerase X / DNA polymerase beta-like, N-terminal domain / Helix-hairpin-helix domain / DNA polymerase X family / DNA polymerase lambda lyase domain superfamily / DNA polymerase family X, binding site / DNA polymerase family X signature. / DNA polymerase family X / DNA polymerase beta, thumb domain / DNA polymerase beta thumb / DNA polymerase, thumb domain superfamily / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Nucleotidyltransferase superfamily
Similarity search - Domain/homology
PHOSPHATE ION / DNA / DNA polymerase lambda
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsKaminski, A.M. / Pedersen, L.C. / Bebenek, K. / Kunkel, T.A. / Chiruvella, K.K. / Ramsden, D.A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIA ES102645 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065070 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA097096 United States
CitationJournal: DNA Repair (Amst) / Year: 2024
Title: DNA polymerase lambda Loop1 variant yields unexpected gain-of-function capabilities in nonhomologous end-joining.
Authors: Kaminski, A.M. / Chiruvella, K.K. / Ramsden, D.A. / Bebenek, K. / Kunkel, T.A. / Pedersen, L.C.
History
DepositionAug 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase lambda
T: DNA (5'-D(*AP*CP*GP*CP*GP*GP*CP*A)-3')
P: DNA (5'-D(P*GP*CP*CP*GP*CP*GP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,0808
Polymers42,8033
Non-polymers2785
Water1,51384
1
A: DNA polymerase lambda
hetero molecules

T: DNA (5'-D(*AP*CP*GP*CP*GP*GP*CP*A)-3')
P: DNA (5'-D(P*GP*CP*CP*GP*CP*GP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)43,0808
Polymers42,8033
Non-polymers2785
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_545x-y,-y-1,-z+1/31
Buried area2560 Å2
ΔGint-39 kcal/mol
Surface area17550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.971, 58.971, 211.248
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA polymerase lambda / / Pol Lambda / DNA polymerase beta-2 / Pol beta2 / DNA polymerase kappa


Mass: 37938.430 Da / Num. of mol.: 1 / Mutation: Ser463-Gln471 deletion and replacement with KGET
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLL / Plasmid: pET28M / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 (DE3)
References: UniProt: Q9UGP5, DNA-directed DNA polymerase, Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases

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DNA chain , 2 types, 2 molecules TP

#2: DNA chain DNA (5'-D(*AP*CP*GP*CP*GP*GP*CP*A)-3')


Mass: 2436.619 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*CP*CP*GP*CP*GP*TP*A)-3')


Mass: 2427.605 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 5 types, 89 molecules

#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#6: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 84 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.48 Å3/Da / Density % sol: 50.35 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: 0.1 M ADA pH 6.5, 1 M ammonium phosphate dibasic

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 12, 2021
RadiationMonochromator: DOUBLE CRYSTAL SI(III) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.05→50 Å / Num. obs: 27862 / % possible obs: 99.9 % / Redundancy: 9.6 % / Biso Wilson estimate: 45.28 Å2 / CC1/2: 0.993 / CC star: 0.998 / Rpim(I) all: 0.025 / Rrim(I) all: 0.078 / Rsym value: 0.073 / Χ2: 0.689 / Net I/σ(I): 30.92
Reflection shellResolution: 2.05→2.09 Å / Redundancy: 7.9 % / Mean I/σ(I) obs: 1.25 / Num. unique obs: 1349 / CC1/2: 0.804 / CC star: 0.944 / Rpim(I) all: 0.313 / Rrim(I) all: 0.931 / Rsym value: 0.874 / Χ2: 0.351 / % possible all: 99.3

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHENIX1.20.1_4487refinement
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.05→41.34 Å / SU ML: 0.2451 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 29.5615
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2586 1368 5.01 %random
Rwork0.2148 25951 --
obs0.217 27319 98.16 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.81 Å2
Refinement stepCycle: LAST / Resolution: 2.05→41.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2244 327 15 84 2670
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01192686
X-RAY DIFFRACTIONf_angle_d1.16673715
X-RAY DIFFRACTIONf_chiral_restr0.0587420
X-RAY DIFFRACTIONf_plane_restr0.0116426
X-RAY DIFFRACTIONf_dihedral_angle_d13.4433806
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.05-2.120.34891280.27912445X-RAY DIFFRACTION94.63
2.12-2.210.29971330.24122476X-RAY DIFFRACTION96.1
2.21-2.310.29791290.2592526X-RAY DIFFRACTION97.07
2.31-2.430.30661330.22442545X-RAY DIFFRACTION97.59
2.43-2.580.29551380.2492588X-RAY DIFFRACTION98.84
2.58-2.780.291340.24712588X-RAY DIFFRACTION99.16
2.78-3.060.30461400.2752624X-RAY DIFFRACTION99.46
3.06-3.50.29731370.21452652X-RAY DIFFRACTION99.68
3.5-4.410.23131440.19072665X-RAY DIFFRACTION99.61
4.41-41.340.22081520.19042842X-RAY DIFFRACTION99.37
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.896953067255-0.306387270445-0.2287895573210.9789473065910.4159531146940.343259434276-0.346824262770.05307867283830.429666264547-0.3161931067810.3728625050340.713093925048-0.531785725978-0.860833837030.0003335924502391.012862254880.1778120614160.1011152583980.8272681219380.07715076138631.01128057652-8.84657553483-8.6735772756123.5368892664
21.28711550898-0.8649946066-0.7411276394391.74092864010.454592108940.6067596461010.0679980141672-0.5620942518290.7009729484510.3869378003670.195433122193-0.354445124088-0.7673205976720.5327004719330.0003809767458310.750711310563-0.155191854103-0.007306296525370.424842865355-0.1229244650650.60750955712218.5661702313-11.120692853223.7684001892
33.583156622671.644745983680.4660292596152.40249609909-0.3620627598892.106790651050.03122138246660.312955846108-0.298582740179-0.2613208163710.00976952942554-0.4435948592440.3969241526570.2993300129764.0365222367E-80.476821809983-0.03419231202160.03125335892940.350759848478-0.03126505002230.38925442124816.1283474747-30.5771401816.86025465869
42.088239894630.302387566717-0.09718155141630.9316837417690.6888670255861.950387853710.0834903616041-0.162593332524-0.05234679788680.2271498108430.03173108486770.5708784924550.379613645794-0.7285658887450.001759167548840.555308898332-0.1697010612210.03756027825240.5095522586120.007122916297180.508816137339-3.55403570327-34.410670649315.5274333532
50.305738505779-0.1044483250180.02206686793860.1996133079220.1319038895420.225048616840.3364406678820.5783772688320.706872460657-0.1293814406010.1789380325380.0183698815927-0.404390629526-0.6269727350647.01192997163E-70.6153598979530.08097353860370.1518944651860.6086787815490.06431592071390.592580669599-14.2053644485-23.218904182138.1376594833
60.5405952124370.4629210208820.4123107699950.3630144653780.3807490325690.3743703091140.01375400759980.344986305610.01702177801480.08251847097340.085991248210.253330120394-0.2132241764140.38370364713-5.62291147865E-80.5338442422890.04357116890540.1122321722930.5503699043390.01177149064620.556540775687-8.33768284739-22.733017138538.0543771947
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain A and resid 250:329AA250 - 3291 - 80
22chain A and resid 330:386AA330 - 38681 - 137
33chain A and resid 387:495AA387 - 495138 - 241
44chain A and resid 496:575AA496 - 575242 - 307
55chain PPC1 - 8
66chain TTB1 - 8

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