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- PDB-1ma7: Crystal structure of Cre site-specific recombinase complexed with... -

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Basic information

Entry
Database: PDB / ID: 1ma7
TitleCrystal structure of Cre site-specific recombinase complexed with a mutant DNA substrate, LoxP-A8/T27
Components
  • (LOXP) x 2
  • CRE RECOMBINASE
KeywordsHYDROLASE / LIGASE/DNA / Cre-Lox site-specific recombination / Specificity of protein-DNA interactions / protein-DNA complex / Tyrosine recombinase / LIGASE-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 / Core-binding (CB) domain / Tyrosine recombinase domain profile. / Core-binding (CB) domain profile. / Phage integrase family / Integrase, catalytic domain / Integrase/recombinase, N-terminal / Integrase-like, catalytic domain superfamily ...Intergrase catalytic core / Tyrosine recombinase, N-terminal domain / hpI Integrase; Chain A / Core-binding (CB) domain / Tyrosine recombinase domain profile. / Core-binding (CB) domain profile. / Phage integrase family / Integrase, catalytic domain / 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 speciesEnterobacteria phage P1 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.3 Å
AuthorsMartin, S.S. / Chu, V.C. / Baldwin, E.P.
Citation
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.P.
#1: 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.
#2: Journal: Nature / Year: 1997
Title: Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse
Authors: Guo, F. / Gopaul, D. / van Duyne, G.D.
#3: Journal: Annu.Rev.Biophys.Biomol.Struct. / Year: 2001
Title: A structural view of cre-loxp site-specific recombination
Authors: van Duyne, G.D.
History
DepositionAug 1, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2003Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Feb 14, 2024Group: 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 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: LOXP
D: LOXP
A: CRE RECOMBINASE
B: CRE RECOMBINASE


Theoretical massNumber of molelcules
Total (without water)99,7574
Polymers99,7574
Non-polymers00
Water5,098283
1
C: LOXP
D: LOXP
A: CRE RECOMBINASE
B: CRE RECOMBINASE

C: LOXP
D: LOXP
A: CRE RECOMBINASE
B: CRE RECOMBINASE


Theoretical massNumber of molelcules
Total (without water)199,5138
Polymers199,5138
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)107.540, 122.240, 180.030
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
DetailsTHE SECOND HALF OF THE TETRAMERIC BIOLOGICAL ASSEMBLY IS GENERATED BY THE CRYSTALLOGRAPHIC TWO-FOLD AXIS: X, -Y, -Z, AND A TRANSLATION OF 0, 1, 1.

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Components

#1: DNA chain LOXP


Mass: 10469.798 Da / Num. of mol.: 1 / Fragment: UPPER STRAND / Mutation: C8A,G27T / Source method: obtained synthetically
#2: DNA chain LOXP


Mass: 10438.788 Da / Num. of mol.: 1 / Fragment: LOWER STRAND / Mutation: C8A,G27T / Source method: obtained synthetically
#3: Protein CRE RECOMBINASE / CRE SITE-SPECIFIC RECOMBINASE


Mass: 39424.047 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage P1 (virus) / Genus: P1-like viruses / 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.: 283 / 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: 2.95 Å3/Da / Density % sol: 58.24 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 5.25
Details: 2,4-methylpentanediol, sodium acetate, calcium chloride, pH 5.25, VAPOR DIFFUSION, HANGING DROP, temperature 294K
Components of the solutions
IDNameCrystal-IDSol-ID
12,4-methylpentanediol11
2sodium acetate11
3CaCl211
4CaCl212
Crystal grow
*PLUS
Temperature: 21 ℃ / Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
124-28 %MPD1reservoirpH5.25
225 mM1reservoir
330 mM1reservoirCaCl2
440 mM1reservoirNaCl

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Jun 28, 2000
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.3→30 Å / Num. all: 54160 / Num. obs: 51997 / % possible obs: 97 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 3.3 % / Biso Wilson estimate: 47 Å2 / Rmerge(I) obs: 0.034 / Net I/σ(I): 21
Reflection shellResolution: 2.3→2.35 Å / Redundancy: 2.9 % / Rmerge(I) obs: 0.275 / Mean I/σ(I) obs: 3.8 / Num. unique all: 3473 / % possible all: 97.7
Reflection
*PLUS
% possible obs: 96.5 % / Num. measured all: 168540
Reflection shell
*PLUS
% possible obs: 97.7 % / Num. unique obs: 3473

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
TNTrefinement
TNTphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: PDB entry 1KBU with mutated basepairs, side chains Arg259, Asp262, and Asp266 omitted

1kbu


Resolution: 2.3→5 Å
Isotropic thermal model: No B value correction in scaling Fcalc to Fobs during refinement. Final R calculation with K=1.2137 B=0.44240 (TNT)
σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.29 2620 -RANDOM
Rwork0.239 ---
all-51364 --
obs-51364 97 %-
Displacement parametersBiso mean: 57 Å2
Refine analyzeLuzzati d res low obs: 5 Å / Luzzati sigma a obs: 0.56 Å
Refinement stepCycle: LAST / Resolution: 2.3→5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5069 1347 0 283 6699
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONt_bond_d0.006
X-RAY DIFFRACTIONt_angle_deg1.174
LS refinement shellResolution: 2.3→2.38 Å /
Num. reflection% reflection
Rfree225 -
obs-92 %
Refinement
*PLUS
Num. reflection obs: 43981 / Num. reflection Rfree: 2383 / Rfactor Rfree: 0.29 / Rfactor Rwork: 0.24
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Type: t_angle_deg / Dev ideal: 1.19
LS refinement shell
*PLUS
Rfactor Rfree: 0.363 / Rfactor Rwork: 0.39

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