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- PDB-1pvq: BASIS FOR A SWITCH IN SUBSTRATE SPECIFICITY: CRYSTAL STRUCTURE OF... -

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Basic information

Entry
Database: PDB / ID: 1pvq
TitleBASIS FOR A SWITCH IN SUBSTRATE SPECIFICITY: CRYSTAL STRUCTURE OF SELECTED VARIANT OF CRE SITE-SPECIFIC RECOMBINASE, LNSGG BOUND TO THE ENGINEERED RECOGNITION SITE LOXM7
Components
  • (DNA 34-MER) x 2
  • Recombinase cre
KeywordsRECOMBINATION/DNA / CRE / Recombinase / DNA / CRE-LOXP RECOMBINATION / RECOMBINATION-DNA COMPLEX
Function / homology
Function and homology information


DNA integration / DNA recombination / DNA binding
Similarity search - Function
: / Intergrase catalytic core / Tyrosine recombinase, N-terminal domain / hpI Integrase; Chain A / Phage integrase family / Core-binding (CB) domain / Core-binding (CB) domain profile. / Integrase, catalytic domain / Tyrosine recombinase domain profile. / Integrase/recombinase, N-terminal ...: / Intergrase catalytic core / Tyrosine recombinase, N-terminal domain / hpI Integrase; Chain A / Phage integrase family / Core-binding (CB) domain / Core-binding (CB) domain profile. / Integrase, catalytic domain / Tyrosine recombinase domain profile. / Integrase/recombinase, N-terminal / Integrase-like, catalytic domain superfamily / DNA breaking-rejoining enzyme, catalytic core / DNA polymerase; domain 1 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
: / : / DNA / DNA (> 10) / Recombinase cre
Similarity search - Component
Biological speciesEscherichia phage P1 (virus)
Escherichia virus P1
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.75 Å
AuthorsBaldwin, E.P. / Martin, S.S. / Abel, J. / Gelato, K.A. / Kim, H. / Schultz, P.G. / Santoro, S.W.
Citation
Journal: Chem.Biol. / Year: 2003
Title: A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water.
Authors: Baldwin, E.P. / Martin, S.S. / Abel, J. / Gelato, K.A. / Kim, H. / Schultz, P.G. / Santoro, S.W.
#1: Journal: Proc.Natl.Acad.Sci.USA / Year: 2002
Title: Directed evolution of the site specificity of Cre recombinase.
Authors: Santoro, S.W. / Schultz, P.G.
#2: Journal: Biochemistry / Year: 2003
Title: Modulation of the active complex assembly and turnover rate by protein-DNA interactions in Cre-LoxP recombination.
Authors: Martin, S.S. / Chu, V.C. / Baldwin, E.
#3: Journal: J.Mol.Biol. / Year: 2002
Title: The order of strand exchanges in Cre-LoxP recombination and its basis suggested by the crystal structure of a Cre-LoxP Holliday junction complex.
Authors: Martin, S.S. / Pulido, E. / Chu, V.C. / Lechner, T.S. / Baldwin, E.P.
History
DepositionJun 28, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 17, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Feb 1, 2012Group: Other
Revision 2.0Apr 7, 2021Group: Advisory / Database references ...Advisory / Database references / Non-polymer description / Source and taxonomy / Structure summary
Category: chem_comp / entity ...chem_comp / entity / entity_src_gen / pdbx_entity_src_syn / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.details / _entity.pdbx_description / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_beg_seq_num / _entity_src_gen.pdbx_end_seq_num / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_seq_type / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.entity_id / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.pdbx_strand_id / _struct_ref_seq.ref_id / _struct_ref_seq.seq_align_beg / _struct_ref_seq.seq_align_end / _struct_ref_seq_dif.align_id / _struct_ref_seq_dif.db_mon_id / _struct_ref_seq_dif.details / _struct_ref_seq_dif.mon_id / _struct_ref_seq_dif.pdbx_auth_seq_num / _struct_ref_seq_dif.pdbx_pdb_strand_id / _struct_ref_seq_dif.pdbx_seq_db_accession_code / _struct_ref_seq_dif.pdbx_seq_db_name / _struct_ref_seq_dif.pdbx_seq_db_seq_num / _struct_ref_seq_dif.seq_num
Revision 2.1Aug 16, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: DNA 34-MER
D: DNA 34-MER
A: Recombinase cre
B: Recombinase cre


Theoretical massNumber of molelcules
Total (without water)99,3544
Polymers99,3544
Non-polymers00
Water1,20767
1
C: DNA 34-MER
D: DNA 34-MER
A: Recombinase cre
B: Recombinase cre

C: DNA 34-MER
D: DNA 34-MER
A: Recombinase cre
B: Recombinase cre


Theoretical massNumber of molelcules
Total (without water)198,7088
Polymers198,7088
Non-polymers00
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_566x,-y+1,-z+11
Unit cell
Length a, b, c (Å)107.410, 121.350, 180.610
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
DetailsThe second part of the tetrameric biological assembly is generated by the two fold axis: x, -y+1, -z+1.

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Components

#1: DNA chain DNA 34-MER


Mass: 10469.798 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: TOP STRAND OF LOXM7 ENGINEERED DNA SUBSTRATE (LOXP(C7/G28,T8/A27,A9/T26)
Source: (synth.) Escherichia virus P1 / References: GenBank: M10145
#2: DNA chain DNA 34-MER


Mass: 10438.787 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: BOTTOM STRAND OF LOXM7 ENGINEERED DNA SUBSTRATE (LOXP(C7/G28,T8/A27,A9/T26)
Source: (synth.) Escherichia virus P1 / References: GenBank: M10494
#3: Protein Recombinase cre


Mass: 39222.805 Da / Num. of mol.: 2 / Mutation: I174L,T258N,R259S,E262G,E266G
Source method: isolated from a genetically manipulated source
Details: SELECTED CRE SITE-SPECIFIC RECOMBINASE MUTANT (I174L,T258N, R259S, E262G, E266G)
Source: (gene. exp.) Escherichia phage P1 (virus) / Genus: P1-like viruses / Gene: cre / Plasmid: PET28B(+) / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21-DE3 / References: UniProt: P06956
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 67 / Source method: isolated from a natural source / Formula: H2O

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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: 2.94 Å3/Da / Density % sol: 58.19 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: Sodium Acetate, Calcium Chloride, 2,4-Methylpentanediol, pH 5.5, VAPOR DIFFUSION, HANGING DROP, temperature 294K
Components of the solutions
IDNameCrystal-IDSol-ID
1Sodium Acetate11
2Calcium Chloride11
32,4-Methylpentanediol11
4H2O11
5Sodium Acetate12
6Calcium Chloride12
72,4-Methylpentanediol12
8H2O12
Crystal grow
*PLUS
Temperature: 21-25 ℃ / Method: vapor diffusion / Details: Martin, S.S., (2002) J.Mol.Biol., 319, 107. / PH range low: 5.5 / PH range high: 5
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
135-45 mg/mlprotein1drop
225 mMsodium acetate1reservoir
340 mM1reservoirNaCl
420 mM1reservoirCaCl2
527 %LMSGG/LoxM71reservoirpH5.5
61
71
81

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL7-1 / Wavelength: 1.08 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jun 12, 2002
RadiationMonochromator: DOUBLE-CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.08 Å / Relative weight: 1
ReflectionResolution: 2.75→90 Å / Num. all: 31227 / Num. obs: 29853 / % possible obs: 95.6 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 3.8 % / Rmerge(I) obs: 0.044 / Net I/σ(I): 17.3
Reflection shellResolution: 2.75→2.85 Å / Rmerge(I) obs: 0.343 / Mean I/σ(I) obs: 3.8 / Num. unique all: 3001 / % possible all: 97.8
Reflection
*PLUS
% possible obs: 96 %
Reflection shell
*PLUS
% possible obs: 98 %

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
TNTrefinement
TNTphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: PDB ENTRY 1KBU

1kbu


Resolution: 2.75→5 Å / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.281 1189 -RANDOM
Rwork0.224 ---
obs0.224 24813 97 %-
Refine analyzeLuzzati d res low obs: 5 Å / Luzzati sigma a obs: 0.27 Å
Refinement stepCycle: LAST / Resolution: 2.75→5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5055 1388 0 67 6510
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONt_bond_d0.006
X-RAY DIFFRACTIONt_angle_deg1.3
Refinement
*PLUS
Lowest resolution: 5 Å
Solvent computation
*PLUS
Displacement parameters
*PLUS

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