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- PDB-8scn: Bst DNA polymerase I Large Fragment mutant F710Y/D598A with 3'-am... -

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Basic information

Entry
Database: PDB / ID: 8scn
TitleBst DNA polymerase I Large Fragment mutant F710Y/D598A with 3'-amino primer, dGTP, and calcium time-resolved 24h (Product State)
Components
  • DNA polymerase I
  • DNA template
  • NP-extended DNA primer
KeywordsREPLICATION/DNA / DNA Polymerase / NP-DNA / Origin of Life / Time-Resolved Crystallography / REPLICATION-DNA complex
Function / homology
Function and homology information


3'-5' exonuclease activity / DNA-templated DNA replication / double-strand break repair / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding
Similarity search - Function
: / DNA polymerase I, ribonuclease H-like domain / DNA polymerase 1 / 3'-5' exonuclease / 3'-5' exonuclease domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain ...: / DNA polymerase I, ribonuclease H-like domain / DNA polymerase 1 / 3'-5' exonuclease / 3'-5' exonuclease domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DIPHOSPHATE / DNA / DNA (> 10) / DNA polymerase I
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsFang, Z. / Lelyveld, V.S. / Szostak, J.W.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Simons Foundation290363 United States
CitationJournal: Science / Year: 2023
Title: Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity.
Authors: Lelyveld, V.S. / Fang, Z. / Szostak, J.W.
History
DepositionApr 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _pdbx_struct_assembly_gen.asym_id_list
Revision 1.2Nov 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase I
B: NP-extended DNA primer
C: DNA template
D: DNA polymerase I
E: NP-extended DNA primer
F: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,35518
Polymers146,1596
Non-polymers1,19712
Water8,053447
1
A: DNA polymerase I
B: NP-extended DNA primer
C: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,96612
Polymers73,0793
Non-polymers8869
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7560 Å2
ΔGint-104 kcal/mol
Surface area27210 Å2
MethodPISA
2
D: DNA polymerase I
E: NP-extended DNA primer
F: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,3896
Polymers73,0793
Non-polymers3103
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6840 Å2
ΔGint-47 kcal/mol
Surface area27370 Å2
MethodPISA
Unit cell
Length a, b, c (Å)93.844, 108.793, 149.905
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 2 molecules AD

#1: Protein DNA polymerase I


Mass: 66073.750 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Gene: DPO1, polA / Production host: Escherichia coli (E. coli) / References: UniProt: D9N168, DNA-directed DNA polymerase

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DNA chain , 2 types, 4 molecules BECF

#2: DNA chain NP-extended DNA primer


Mass: 3069.032 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA template


Mass: 3936.575 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 459 molecules

#4: Chemical ChemComp-DPO / DIPHOSPHATE


Mass: 173.943 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: O7P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: SO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 447 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.62 Å3/Da / Density % sol: 53.01 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 2 M ammonium sulphate, 0.1 M MES pH 5.3-6.0, 20 mM CaCl2, 5% MPD
PH range: 5.3-6.0

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Data collection

DiffractionMean temperature: 99 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 1, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.3→50 Å / Num. obs: 66403 / % possible obs: 96.7 % / Redundancy: 7.3 % / Biso Wilson estimate: 28.16 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.093 / Rpim(I) all: 0.037 / Rrim(I) all: 0.1 / Χ2: 0.946 / Net I/σ(I): 21.7
Reflection shellResolution: 2.3→2.34 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.483 / Mean I/σ(I) obs: 4.9 / Num. unique obs: 3372 / CC1/2: 0.924 / CC star: 0.98 / Rpim(I) all: 0.194 / Rrim(I) all: 0.521 / Χ2: 0.814 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.3→44.11 Å / SU ML: 0.2782 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 23.9647
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2428 3280 4.96 %
Rwork0.1856 62913 -
obs0.1884 66193 96.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 32.12 Å2
Refinement stepCycle: LAST / Resolution: 2.3→44.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9292 900 60 447 10699
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.007610514
X-RAY DIFFRACTIONf_angle_d0.900314402
X-RAY DIFFRACTIONf_chiral_restr0.05021610
X-RAY DIFFRACTIONf_plane_restr0.00521704
X-RAY DIFFRACTIONf_dihedral_angle_d22.62974044
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.340.29061460.2192713X-RAY DIFFRACTION97.08
2.34-2.370.2661430.20292790X-RAY DIFFRACTION100
2.37-2.410.30021390.1972818X-RAY DIFFRACTION99.97
2.41-2.450.26991400.20732785X-RAY DIFFRACTION99.93
2.45-2.50.27971480.19982819X-RAY DIFFRACTION100
2.5-2.550.27321580.19612776X-RAY DIFFRACTION100
2.55-2.60.2721340.19942828X-RAY DIFFRACTION100
2.6-2.650.33421580.20792803X-RAY DIFFRACTION100
2.65-2.720.30181380.20752830X-RAY DIFFRACTION100
2.72-2.780.28311540.21242785X-RAY DIFFRACTION99.97
2.78-2.860.26751410.20492823X-RAY DIFFRACTION99.97
2.86-2.940.28071390.21142840X-RAY DIFFRACTION99.97
2.94-3.040.26911480.2072825X-RAY DIFFRACTION100
3.04-3.150.28071550.2032810X-RAY DIFFRACTION99.97
3.15-3.270.23021450.1952812X-RAY DIFFRACTION99.97
3.27-3.420.26371540.18532830X-RAY DIFFRACTION99.87
3.42-3.60.22561520.17762799X-RAY DIFFRACTION98.5
3.6-3.830.2529930.2041575X-RAY DIFFRACTION55.92
3.83-4.120.22981000.17881941X-RAY DIFFRACTION68.03
4.12-4.540.20511400.15012893X-RAY DIFFRACTION99.9
4.54-5.190.20181630.15272863X-RAY DIFFRACTION99.97
5.19-6.540.21971360.17652930X-RAY DIFFRACTION100
6.54-44.110.17821560.16493025X-RAY DIFFRACTION99.1

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