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- PDB-4ahc: Crystal Structure of an Evolved Replicating DNA Polymerase -

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Basic information

Entry
Database: PDB / ID: 4ahc
TitleCrystal Structure of an Evolved Replicating DNA Polymerase
ComponentsDNA POLYMERASE
KeywordsTRANSFERASE / B FAMILY POLYMERASE / APOENZYME
Function / homology
Function and homology information


DNA-templated DNA replication / endonuclease activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding
Similarity search - Function
B family DNA polymerase, thumb domain / DNA Polymerase; Chain A, domain 1 / DNA Polymerase, chain B, domain 1 / B family DNA polymerase, finger domain / Palm domain of DNA polymerase / B family DNA polymerase, palm domain / : / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain ...B family DNA polymerase, thumb domain / DNA Polymerase; Chain A, domain 1 / DNA Polymerase, chain B, domain 1 / B family DNA polymerase, finger domain / Palm domain of DNA polymerase / B family DNA polymerase, palm domain / : / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / Topoisomerase I; Chain A, domain 4 / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H-like superfamily/Ribonuclease H / Nucleotidyltransferase; domain 5 / Helix Hairpins / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / Alpha-Beta Complex / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Biological speciesPYROCOCCUS FURIOSUS (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsWynne, S.A. / Holliger, P. / Leslie, A.G.W.
Citation
Journal: Plos One / Year: 2013
Title: Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA.
Authors: Wynne, S.A. / Pinheiro, V.B. / Holliger, P. / Leslie, A.G.
#1: Journal: J.Am.Chem.Soc. / Year: 2010
Title: Cydna: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase.
Authors: Ramsay, N. / Jemth, A. / Brown, A. / Crampton, N. / Dear, P. / Holliger, P.
History
DepositionFeb 6, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 7, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2013Group: Database references
Revision 1.2Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_ncs_dom_lim / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA POLYMERASE
B: DNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,5956
Polymers180,2262
Non-polymers3684
Water5,296294
1
A: DNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,2973
Polymers90,1131
Non-polymers1842
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: DNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,2973
Polymers90,1131
Non-polymers1842
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)78.438, 197.862, 78.438
Angle α, β, γ (deg.)90.00, 108.39, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Ens-ID: 1 / Refine code: 6

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11METMETASPASPAA1 - 441 - 44
21METMETASPASPBB1 - 441 - 44
12ILEILELEULEUAA54 - 7654 - 76
22ILEILELEULEUBB54 - 7654 - 76
13PROPROPROPROAA79 - 9079 - 90
23PROPROPROPROBB79 - 9079 - 90
14ASPASPGLYGLYAA92 - 12592 - 125
24ASPASPGLYGLYBB92 - 12592 - 125
15LYSLYSTHRTHRAA136 - 144136 - 144
25LYSLYSTHRTHRBB136 - 144136 - 144
16GLYGLYASPASPAA155 - 164155 - 164
26GLYGLYASPASPBB155 - 164155 - 164
17VALVALVALVALAA170 - 181170 - 181
27VALVALVALVALBB170 - 181170 - 181
18VALVALILEILEAA183 - 198183 - 198
28VALVALILEILEBB183 - 198183 - 198
19ILEILEASPASPAA205 - 212205 - 212
29ILEILEASPASPBB205 - 212205 - 212
110PHEPHEMETMETAA216 - 241216 - 241
210PHEPHEMETMETBB216 - 241216 - 241
111VALVALILEILEAA250 - 264250 - 264
211VALVALILEILEBB250 - 264250 - 264
112LEULEUPHEPHEAA270 - 283270 - 283
212LEULEUPHEPHEBB270 - 283270 - 283
113ASPASPGLUGLUAA315 - 363315 - 363
213ASPASPGLUGLUBB315 - 363315 - 363
114GLUGLUTYRTYRAA366 - 403366 - 403
214GLUGLUTYRTYRBB366 - 403366 - 403
115ILEILEGLUGLUAA413 - 459413 - 459
215ILEILEGLUGLUBB413 - 459413 - 459
116ARGARGTRPTRPAA461 - 526461 - 526
216ARGARGTRPTRPBB461 - 526461 - 526
117PHEPHETYRTYRAA535 - 584535 - 584
217PHEPHETYRTYRBB535 - 584535 - 584
118GLYGLYARGARGAA587 - 607587 - 607
218GLYGLYARGARGBB587 - 607587 - 607
119ARGARGILEILEAA626 - 631626 - 631
219ARGARGILEILEBB626 - 631626 - 631

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(0.316318, 0.001265, -0.948652), (-0.000193, -0.999999, -0.001397), (-0.948653, 0.000625, -0.316318)-45.41342, 6.05532, -62.9024

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Components

#1: Protein DNA POLYMERASE / PFU POLYMERASE


Mass: 90113.172 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PYROCOCCUS FURIOSUS (archaea) / Plasmid: PT7-SC / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): STRATAGENE CODON PLUS - RIL / References: UniProt: P61875, DNA-directed DNA polymerase
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 294 / Source method: isolated from a natural source / Formula: H2O
Compound detailsENGINEERED RESIDUE IN CHAIN A, VAL 93 TO GLN ENGINEERED RESIDUE IN CHAIN A, ASP 141 TO ALA ...ENGINEERED RESIDUE IN CHAIN A, VAL 93 TO GLN ENGINEERED RESIDUE IN CHAIN A, ASP 141 TO ALA ENGINEERED RESIDUE IN CHAIN A, GLU 143 TO ALA ENGINEERED RESIDUE IN CHAIN A, VAL 337 TO ILE ENGINEERED RESIDUE IN CHAIN A, GLU 399 TO ASP ENGINEERED RESIDUE IN CHAIN A, ASN 400 TO ASP ENGINEERED RESIDUE IN CHAIN A, ARG 407 TO ILE ENGINEERED RESIDUE IN CHAIN A, TYR 546 TO HIS ENGINEERED RESIDUE IN CHAIN B, VAL 93 TO GLN ENGINEERED RESIDUE IN CHAIN B, ASP 141 TO ALA ENGINEERED RESIDUE IN CHAIN B, GLU 143 TO ALA ENGINEERED RESIDUE IN CHAIN B, VAL 337 TO ILE ENGINEERED RESIDUE IN CHAIN B, GLU 399 TO ASP ENGINEERED RESIDUE IN CHAIN B, ASN 400 TO ASP ENGINEERED RESIDUE IN CHAIN B, ARG 407 TO ILE ENGINEERED RESIDUE IN CHAIN B, TYR 546 TO HIS
Has protein modificationY
Sequence detailsTHIS DNA POLYMERASE HAS MUTATIONS INTRODUCED BY DIRECTED EVOLUTION, TO ENABLE IT TO INCORPORATE CY- ...THIS DNA POLYMERASE HAS MUTATIONS INTRODUCED BY DIRECTED EVOLUTION, TO ENABLE IT TO INCORPORATE CY-DYE LABELLED NUCLEOTIDES INTO THE NEW DNA STRAND. MUTATIONS AT V93Q, D141A, E143A, V337I, E399D, N400D, R407I AND Y546H

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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: 3.21 Å3/Da / Density % sol: 61.65 % / Description: NONE
Crystal growpH: 6.5
Details: 0.08M SODIUM CACODYLATE PH 6.5 0.14M MAGNESIUM ACETATE 12% PEG 8K 20% GLYCEROL

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-4 / Wavelength: 0.9334
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Aug 30, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9334 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.546
11L, -K, H20.454
ReflectionResolution: 2.4→41.2 Å / Num. obs: 78466 / % possible obs: 89.1 % / Observed criterion σ(I): -5 / Redundancy: 2.7 % / Rmerge(I) obs: 0.08 / Net I/σ(I): 9.6
Reflection shellResolution: 2.4→2.46 Å / Redundancy: 2.3 % / Rmerge(I) obs: 0.47 / Mean I/σ(I) obs: 2.1 / % possible all: 83

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Processing

Software
NameVersionClassification
REFMAC5.7.0002refinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1TGO

1tgo


Resolution: 2.4→98.95 Å / Cor.coef. Fo:Fc: 0.935 / Cor.coef. Fo:Fc free: 0.925 / SU B: 5.733 / SU ML: 0.141 / Cross valid method: THROUGHOUT / ESU R: 0.076 / ESU R Free: 0.05 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.22936 3805 4.9 %RANDOM
Rwork0.20626 ---
obs0.20739 74622 88.8 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 41.479 Å2
Baniso -1Baniso -2Baniso -3
1--15.31 Å20 Å22.3 Å2
2--33.42 Å20 Å2
3----18.11 Å2
Refinement stepCycle: LAST / Resolution: 2.4→98.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11863 0 24 294 12181
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.01912150
X-RAY DIFFRACTIONr_bond_other_d0.0010.028664
X-RAY DIFFRACTIONr_angle_refined_deg0.7611.98116375
X-RAY DIFFRACTIONr_angle_other_deg0.7153.00121099
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.38151439
X-RAY DIFFRACTIONr_dihedral_angle_2_deg28.23923.989564
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.139152301
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.7731577
X-RAY DIFFRACTIONr_chiral_restr0.0470.21771
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.02113140
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022457
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 7128 / Refine-ID: X-RAY DIFFRACTION

Dom-IDAuth asym-IDTypeRms dev position (Å)Weight position
1Aloose positional0.265
2Bloose positional0.265
1Aloose thermal1.6810
2Bloose thermal1.6810
LS refinement shellResolution: 2.4→2.462 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.341 221 -
Rwork0.286 4988 -
obs--80.11 %

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