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- PDB-1lws: Crystal structure of the intein homing endonuclease PI-SceI bound... -

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Basic information

Entry
Database: PDB / ID: 1lws
TitleCrystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence
Components
  • ENDONUCLEASE PI-SCEI
  • PI-SceI DNA recognition region bottom strand
  • PI-SceI DNA recognition region top strand
KeywordsHYDROLASE/DNA / homing endonuclease / intein / protein-DNA complex / endonuclease / HYDROLASE-DNA COMPLEX
Function / homology
Function and homology information


Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / endosomal lumen acidification / vacuolar proton-transporting V-type ATPase, V1 domain / proton-transporting V-type ATPase complex / protein metabolic process / intein-mediated protein splicing ...Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / endosomal lumen acidification / vacuolar proton-transporting V-type ATPase, V1 domain / proton-transporting V-type ATPase complex / protein metabolic process / intein-mediated protein splicing / intron homing / fungal-type vacuole membrane / vacuolar proton-transporting V-type ATPase complex / vacuolar acidification / H+-transporting two-sector ATPase / proton transmembrane transport / phagocytic vesicle / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / endonuclease activity / Hydrolases; Acting on ester bonds / Golgi membrane / mRNA binding / DNA binding / ATP binding
Similarity search - Function
Homing endonuclease PI-Sce / Homing endonuclease / Hom-end-associated Hint / Hom_end-associated Hint / Endonuclease - Pi-scei; Chain A, domain 1 / Hedgehog/Intein (Hint) domain / Intein / Homing endonucleases / Endonuclease I-creI / Intein DOD homing endonuclease ...Homing endonuclease PI-Sce / Homing endonuclease / Hom-end-associated Hint / Hom_end-associated Hint / Endonuclease - Pi-scei; Chain A, domain 1 / Hedgehog/Intein (Hint) domain / Intein / Homing endonucleases / Endonuclease I-creI / Intein DOD homing endonuclease / Intein DOD-type homing endonuclease domain profile. / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Homing endonuclease / Hint domain superfamily / V-type ATP synthase catalytic alpha chain / ATPsynthase alpha/beta subunit, N-terminal extension / ATPsynthase alpha/beta subunit N-term extension / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / ATP synthase subunit alpha, N-terminal domain-like superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain / ATP synthase alpha/beta family, beta-barrel domain / ATPase, alpha/beta subunit, nucleotide-binding domain, active site / ATP synthase alpha and beta subunits signature. / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / Beta Complex / Roll / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / V-type proton ATPase catalytic subunit A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 3.5 Å
AuthorsMoure, C.M. / Gimble, F.S. / Quiocho, F.A.
Citation
Journal: Nat.Struct.Biol. / Year: 2002
Title: Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence.
Authors: Moure, C.M. / Gimble, F.S. / Quiocho, F.A.
#1: Journal: Cell(Cambridge,Mass.) / Year: 1997
Title: Crystal structure of PI-SceI, a homing endonuclease with protein splicing activity
Authors: Duan, X. / Gimble, F.S. / Quiocho, F.A.
History
DepositionJun 3, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 27, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: PI-SceI DNA recognition region top strand
C: PI-SceI DNA recognition region bottom strand
A: ENDONUCLEASE PI-SCEI
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,2315
Polymers74,1513
Non-polymers802
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)123.000, 123.000, 211.700
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522

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Components

#1: DNA chain PI-SceI DNA recognition region top strand


Mass: 11599.467 Da / Num. of mol.: 1 / Source method: obtained synthetically
#2: DNA chain PI-SceI DNA recognition region bottom strand


Mass: 11172.185 Da / Num. of mol.: 1 / Source method: obtained synthetically
#3: Protein ENDONUCLEASE PI-SCEI / PI-SceI / VMA-derived endonuclease / VDE / SCE VMA intein


Mass: 51379.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: VMA1 / Plasmid: PT7PI-SceI / Production host: Escherichia coli (E. coli) / Strain (production host): DL41 (DE3)
References: UniProt: P17255, Hydrolases; Acting on ester bonds
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

CrystalDensity Matthews: 3.08 Å3/Da / Density % sol: 60.06 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: PEG 200, na hepes, calcium chloride, pH 7.5, VAPOR DIFFUSION, HANGING DROP, temperature 291K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 20011
2na hepes11
3CaCl211
4CaCl212
5na hepes12
Crystal grow
*PLUS
Temperature: 18 ℃ / pH: 8
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
16 mg/mlprotein1drop
260 mM1dropKCl
30.5 mMEDTA1drop
42.5 mM1dropCaCl2
51 mMTCEP1drop
610 mMTris-HCl1droppH8.
725 %(v/v)PEG2001reservoir
80.2 M1reservoirCaCl2
90.1 Msodium HEPES1reservoirpH7.5

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
12001
21
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONAPS 14-BM-D10.9792, 0.9789, 0.9611
SYNCHROTRONAPS 14-BM-C21
DetectorType: ADSC QUANTUM 4 / Detector: CCD
RadiationProtocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.97921
20.97891
30.96111
411
ReflectionResolution: 3.5→30 Å / Num. all: 12539 / Num. obs: 12539 / % possible obs: 98.6 % / Observed criterion σ(I): -3 / Redundancy: 9 % / Rmerge(I) obs: 0.059 / Rsym value: 0.059 / Net I/σ(I): 18.1
Reflection shellResolution: 3.5→3.63 Å / Rmerge(I) obs: 0.201 / Num. unique all: 1194 / Rsym value: 0.201 / % possible all: 96.8
Reflection
*PLUS
Lowest resolution: 25 Å / Num. measured all: 112701 / Rmerge(I) obs: 0.059
Reflection shell
*PLUS
% possible obs: 96.8 % / Rmerge(I) obs: 0.201

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MAD / Resolution: 3.5→24.92 Å / Rfactor Rfree error: 0.012
Isotropic thermal model: group for protein atoms, overall for DNA
Cross valid method: THROUGHOUT / σ(F): 2 / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.31 622 5.1 %RANDOM
Rwork0.287 ---
all0.293 12418 --
obs-11584 97.6 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 28.1355 Å2 / ksol: 0.210571 e/Å3
Displacement parametersBiso mean: 69.6 Å2
Baniso -1Baniso -2Baniso -3
1-10.91 Å211.77 Å20 Å2
2--10.91 Å20 Å2
3----21.83 Å2
Refine analyzeLuzzati coordinate error free: 0.63 Å / Luzzati sigma a free: 0.89 Å
Refinement stepCycle: LAST / Resolution: 3.5→24.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3360 1394 2 0 4756
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_angle_deg1.6
X-RAY DIFFRACTIONc_dihedral_angle_d22.4
X-RAY DIFFRACTIONc_improper_angle_d1.25
X-RAY DIFFRACTIONc_mcbond_it8.581.5
X-RAY DIFFRACTIONc_mcangle_it13.32
X-RAY DIFFRACTIONc_scbond_it7.642
X-RAY DIFFRACTIONc_scangle_it11.132.5
LS refinement shellResolution: 3.5→3.66 Å / Rfactor Rfree error: 0.043 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.3945 124 4.3 %
Rwork0.3719 1881 -
obs--96.8 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DNA-RNA_REP.PARAMDNA-RNA.TOP
X-RAY DIFFRACTION3ION.PARAMION.TOP
Refinement
*PLUS
Lowest resolution: 25 Å / % reflection Rfree: 5 % / Rfactor all: 0.293 / Rfactor Rfree: 0.31 / Rfactor Rwork: 0.287
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_deg1.678
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.4
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.25
LS refinement shell
*PLUS
Rfactor Rfree: 0.3945 / Rfactor Rwork: 0.3719

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