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

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Basic information

Entry
Database: PDB / ID: 1lwt
TitleCrystal structure of the intein homing endonuclease PI-SceI bound to its substrate DNA (Ca2+ free)
Components
  • ENDONUCLEASE PI-SCEI
  • PI-SceI DNA substrate bottom strand
  • PI-SceI DNA substrate top strand
KeywordsHYDROLASE/DNA / HOMING ENDONUCLEASE / PROTEIN-DNA COMPLEX / INTEIN / 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 / vacuolar proton-transporting V-type ATPase, V1 domain / endosomal lumen acidification / proton-transporting V-type ATPase complex / intron homing / intein-mediated protein splicing ...Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / vacuolar proton-transporting V-type ATPase, V1 domain / endosomal lumen acidification / proton-transporting V-type ATPase complex / intron homing / intein-mediated protein splicing / vacuolar proton-transporting V-type ATPase complex / vacuolar acidification / fungal-type vacuole membrane / proton-transporting ATPase activity, rotational mechanism / H+-transporting two-sector ATPase / ATP metabolic process / proton transmembrane transport / 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. / Homing endonuclease / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily / V-type ATP synthase catalytic alpha chain / ATPsynthase alpha/beta subunit, N-terminal extension / ATPsynthase alpha/beta subunit barrel-sandwich domain / : / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / C-terminal domain of V and A type ATP synthase / 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 / MOLECULAR REPLACEMENT / Resolution: 3.2 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
Revision 1.3Oct 9, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / struct_conn / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: PI-SceI DNA substrate top strand
C: PI-SceI DNA substrate bottom strand
A: ENDONUCLEASE PI-SCEI


Theoretical massNumber of molelcules
Total (without water)74,1513
Polymers74,1513
Non-polymers00
Water27015
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)45.200, 87.400, 214.800
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: DNA chain PI-SceI DNA substrate top strand


Mass: 11599.467 Da / Num. of mol.: 1 / Source method: obtained synthetically
#2: DNA chain PI-SceI DNA substrate 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O
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: 2.83 Å3/Da / Density % sol: 56.49 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 5.8
Details: PEG 3550, na citrate, ammonium acetate, pH 5.8, VAPOR DIFFUSION, HANGING DROP, temperature 291K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 355011
2na citrate11
3ammonium acetate11
4na citrate12
Crystal grow
*PLUS
Temperature: 18 ℃
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
16 mg/mlprotein1drop
220 %(w/v)PEG33501reservoir
30.2 M1reservoir
42.5 %(w/v)sucrose1reservoir
50.1 Msodium citrate1reservoirpH5.8

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Data collection

DiffractionMean temperature: 200 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 14-BM-C / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Nov 27, 2000
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.2→30 Å / Num. all: 12363 / Num. obs: 12363 / % possible obs: 83.6 % / Observed criterion σ(I): -3 / Redundancy: 3.9 % / Rmerge(I) obs: 0.082 / Rsym value: 0.082 / Net I/σ(I): 11.9
Reflection shellResolution: 3.2→3.3 Å / Rmerge(I) obs: 0.193 / Num. unique all: 999 / Rsym value: 0.193 / % possible all: 69.8
Reflection
*PLUS
Lowest resolution: 25 Å / Num. measured all: 117632 / Rmerge(I) obs: 0.082
Reflection shell
*PLUS
% possible obs: 69.9 % / Rmerge(I) obs: 0.193

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1LWS

1lws


Resolution: 3.2→24.49 Å / Rfactor Rfree error: 0.012 / Isotropic thermal model: isotropic / Cross valid method: THROUGHOUT / σ(F): 2 / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.287 546 4.5 %RANDOM
Rwork0.242 ---
all0.248 11779 --
obs-11597 82.4 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 10 Å2 / ksol: 0.18428 e/Å3
Displacement parametersBiso mean: 36.7 Å2
Baniso -1Baniso -2Baniso -3
1-9.535 Å2-25.273 Å215.737 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyzeLuzzati coordinate error free: 0.53 Å / Luzzati sigma a free: 0.59 Å
Refinement stepCycle: LAST / Resolution: 3.2→24.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3539 1511 0 15 5065
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_angle_deg1.8
X-RAY DIFFRACTIONc_dihedral_angle_d23.5
X-RAY DIFFRACTIONc_improper_angle_d1.24
X-RAY DIFFRACTIONc_mcbond_it1.591.5
X-RAY DIFFRACTIONc_mcangle_it2.82
X-RAY DIFFRACTIONc_scbond_it1.792
X-RAY DIFFRACTIONc_scangle_it2.792.5
LS refinement shellResolution: 3.2→3.35 Å / Rfactor Rfree error: 0.4 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.353 54 4.1 %
Rwork0.301 1639 -
obs--71.3 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DNA-RNA_REP.PARAMDNA-RNA.TOP
X-RAY DIFFRACTION3WATER.PARAMWATER.TOP
Refinement
*PLUS
Highest resolution: 3.2 Å / Lowest resolution: 25 Å / % reflection Rfree: 5 % / Rfactor all: 0.248 / Rfactor Rfree: 0.287 / Rfactor Rwork: 0.242
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_deg1.712
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg23.5
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.24
LS refinement shell
*PLUS
Rfactor Rfree: 0.353 / Rfactor Rwork: 0.301

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