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- PDB-1p4e: Flpe W330F mutant-DNA Holliday Junction Complex -

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Basic information

Entry
Database: PDB / ID: 1p4e
TitleFlpe W330F mutant-DNA Holliday Junction Complex
Components
  • (Recombinase FLP ...) x 2
  • 33-MER
  • 5'-D(*TP*AP*AP*GP*TP*TP*CP*CP*TP*AP*TP*TP*C)-3'
  • 5'-D(*TP*TP*TP*AP*AP*AP*AP*GP*AP*AP*TP*AP*GP*GP*AP*AP*CP*TP*TP*C)-3'
KeywordsDNA BINDING PROTEIN/RECOMBINATION/DNA / Flp / Holliday junction / site-specific recombination / W330 / Flpe / DNA BINDING PROTEIN-RECOMBINATION-DNA COMPLEX
Function / homology
Function and homology information


plasmid recombination / site-specific recombinase activity / DNA binding, bending / single-stranded DNA binding / double-stranded DNA binding
Similarity search - Function
FLP Recombinase, lambda integrase-like, N-terminal domain (domain 1) / Recombinase Flp protein, C-terminal / Recombinase Flp protein, N-terminal / Recombinase Flp protein N-terminus / Recombinase Flp protein / Flp-type tyrosine recombinase domain profile. / Intergrase catalytic core / hpI Integrase; Chain A / Integrase-like, catalytic domain superfamily / DNA breaking-rejoining enzyme, catalytic core ...FLP Recombinase, lambda integrase-like, N-terminal domain (domain 1) / Recombinase Flp protein, C-terminal / Recombinase Flp protein, N-terminal / Recombinase Flp protein N-terminus / Recombinase Flp protein / Flp-type tyrosine recombinase domain profile. / Intergrase catalytic core / hpI Integrase; Chain A / Integrase-like, catalytic domain superfamily / DNA breaking-rejoining enzyme, catalytic core / GMP Synthetase; Chain A, domain 3 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHONATE / DNA / DNA (> 10) / Site-specific recombinase Flp
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.7 Å
AuthorsRice, P.A. / Chen, Y.
CitationJournal: J.Biol.Chem. / Year: 2003
Title: The role of the conserved Trp330 in Flp-mediated recombination. Functional and structural analysis
Authors: Rice, P.A. / Chen, Y.
History
DepositionApr 23, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2003Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 16, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.5Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.6Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature
Remark 999 The sequence in the database is that of Flp. For this experiment, it is Flpe, a mutant of Flp. ... The sequence in the database is that of Flp. For this experiment, it is Flpe, a mutant of Flp. Flpe differs from Flp with four point mutations at 2, 33, 108 and 294.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: 5'-D(*TP*AP*AP*GP*TP*TP*CP*CP*TP*AP*TP*TP*C)-3'
I: 5'-D(*TP*TP*TP*AP*AP*AP*AP*GP*AP*AP*TP*AP*GP*GP*AP*AP*CP*TP*TP*C)-3'
F: 5'-D(*TP*AP*AP*GP*TP*TP*CP*CP*TP*AP*TP*TP*C)-3'
J: 5'-D(*TP*TP*TP*AP*AP*AP*AP*GP*AP*AP*TP*AP*GP*GP*AP*AP*CP*TP*TP*C)-3'
G: 33-MER
H: 33-MER
A: Recombinase FLP protein
B: Recombinase FLP protein
C: Recombinase FLP protein
D: Recombinase FLP protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,19011
Polymers238,11010
Non-polymers801
Water4,792266
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)79.760, 116.715, 142.053
Angle α, β, γ (deg.)90.00, 97.64, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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DNA chain , 3 types, 6 molecules EFIJGH

#1: DNA chain 5'-D(*TP*AP*AP*GP*TP*TP*CP*CP*TP*AP*TP*TP*C)-3'


Mass: 3916.571 Da / Num. of mol.: 2 / Source method: obtained synthetically
#2: DNA chain 5'-D(*TP*TP*TP*AP*AP*AP*AP*GP*AP*AP*TP*AP*GP*GP*AP*AP*CP*TP*TP*C)-3'


Mass: 6165.042 Da / Num. of mol.: 2 / Source method: obtained synthetically
#3: DNA chain 33-MER


Mass: 10126.570 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: 2PO represents 3'-phosphate which attached to C13 of chain H

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Recombinase FLP ... , 2 types, 4 molecules ABCD

#4: Protein Recombinase FLP protein / Protein Able


Mass: 49383.395 Da / Num. of mol.: 2 / Fragment: Flpe / Mutation: W330F
Source method: isolated from a genetically manipulated source
Details: C-terminal (His)6 tag via a glycine linker
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: FLP1 / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star (DE3) pLysS / References: UniProt: P03870
#5: Protein Recombinase FLP protein / Protein Able


Mass: 49463.375 Da / Num. of mol.: 2 / Fragment: Flpe / Mutation: W330F
Source method: isolated from a genetically manipulated source
Details: Posttranslational modification of TYR343 to PTR. This modification is a result of covalent binding to DNA.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: FLP1 / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star (DE3) pLysS / References: UniProt: P03870

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Non-polymers , 2 types, 267 molecules

#6: Chemical ChemComp-2PO / PHOSPHONATE


Mass: 79.980 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: HO3P / Details: 3'-phosphate
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 266 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 2.91 Å3/Da / Density % sol: 57.44 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: PEG 5KMME, Hepes, xylitol, sodium chloride, calcium chloride, pH 7.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 500011
2H2O11
3xylitol11
4sodium chlorid11
5calcium chloride11
6PEG 500012
7calcium chloride12
8H2O12
Crystal grow
*PLUS
pH: 8
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
14 mMDNA substrate1drop
2120 nmprotein1drop
325 mMtaps1droppH8.0
4200 mM1dropNaCl
512 %glycerol1drop
62 mMEDTA1drop
71 mg/mlbovine serum albumin1drop
820 mMHEPES1reservoirpH7.0
911 %PEG5000 MME1reservoir
1020 %xylitol1reservoir
11100 mM1reservoirNaCl
122 mMdithiothreitol1reservoir
1310 mM1reservoirCaCl2

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 14-BM-C / Wavelength: 0.9 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Mar 26, 2001
RadiationMonochromator: Ge 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.7→22 Å / Num. all: 68765 / Num. obs: 68765 / % possible obs: 96.1 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 42.1 Å2 / Rmerge(I) obs: 0.043
Reflection shellResolution: 2.7→2.8 Å / % possible all: 90.9
Reflection
*PLUS
Lowest resolution: 22 Å

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Processing

Software
NameVersionClassification
CNS1.1refinement
DENZOdata reduction
SCALEPACKdata scaling
CNS1.1phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: Flpe-DNA complex, pdb. 1M6X

1m6x


Resolution: 2.7→21.98 Å / Rfactor Rfree error: 0.003 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
Details: CHAIN B IS THE MOST WELL-ORDERED protein monomer. The crystal diffraction was anisotropic (2.7, 3.0, 2.9 along the three axes). The distance between T14 H and C13 H is 3.45 angstrom.
RfactorNum. reflection% reflectionSelection details
Rfree0.275 6513 10.1 %RANDOM
Rwork0.236 ---
all-70806 --
obs-64397 90.8 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 32.205 Å2 / ksol: 0.273014 e/Å3
Displacement parametersBiso mean: 79 Å2
Baniso -1Baniso -2Baniso -3
1--15.62 Å20 Å21.42 Å2
2--16.55 Å20 Å2
3----0.93 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.47 Å0.4 Å
Luzzati d res low-5 Å
Luzzati sigma a0.62 Å0.58 Å
Refinement stepCycle: LAST / Resolution: 2.7→21.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13181 2619 4 266 16070
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d20.2
X-RAY DIFFRACTIONc_improper_angle_d0.98
X-RAY DIFFRACTIONc_mcbond_it1.331.5
X-RAY DIFFRACTIONc_mcangle_it2.382
X-RAY DIFFRACTIONc_scbond_it3.682
X-RAY DIFFRACTIONc_scangle_it4.722.5
LS refinement shellResolution: 2.7→2.87 Å / Rfactor Rfree error: 0.013 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.399 932 10.3 %
Rwork0.378 8152 -
obs--77.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DNA-RNA_REP_NOPUCKERS.PARAMDNA-RNA.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER.TOP
Refinement
*PLUS
Highest resolution: 2.7 Å / Lowest resolution: 22 Å / % reflection Rfree: 10 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_deg1.21
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg20.2
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.98

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