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HEADER HYDROLASE 27-APR-05 1ZI6
TITLE CRYSTAL STRUCTURE ANALYSIS OF THE DIENELACTONE HYDROLASE
TITLE 2 (C123S) MUTANT- 1.7 A
COMPND MOL_ID: 1;
COMPND 2 MOLECULE: CARBOXYMETHYLENEBUTENOLIDASE;
COMPND 3 CHAIN: A;
COMPND 4 SYNONYM: DIENELACTONE HYDROLASE;
COMPND 5 EC: 3.1.1.45;
COMPND 6 ENGINEERED: YES;
COMPND 7 MUTATION: YES
SOURCE MOL_ID: 1;
SOURCE 2 ORGANISM_SCIENTIFIC: PSEUDOMONAS PUTIDA;
SOURCE 3 ORGANISM_COMMON: BACTERIA;
SOURCE 4 GENE: CLCD;
SOURCE 5 EXPRESSION_SYSTEM: ESCHERICHIA COLI;
SOURCE 6 EXPRESSION_SYSTEM_COMMON: BACTERIA;
SOURCE 7 EXPRESSION_SYSTEM_STRAIN: DH5ALPHA;
SOURCE 8 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID;
SOURCE 9 EXPRESSION_SYSTEM_PLASMID: PCY76
KEYWDS ALPHA AND BETA PROTEINS, 3-D STRUCTURE, SERINE ESTERASE,
KEYWDS 2 HYDROLASE, AROMATIC HYDROCARBONS, CATABOLISM, PLASMID
EXPDTA X-RAY DIFFRACTION
AUTHOR H.-K.KIM,J.-W.LIU,P.D.CARR,D.L.OLLIS
REVDAT 1 05-JUL-05 1ZI6 0
JRNL AUTH H.-K.KIM,J.-W.LIU,P.D.CARR,D.L.OLLIS
JRNL TITL FOLLOWING DIRECTED EVOLUTION WITH CRYSTALLOGRAPHY:
JRNL TITL 2 STRUCTURAL CHANGES OBSERVED IN CHANGING THE
JRNL TITL 3 SUBSTRATE SPECIFICITY OF DIENELACTONE HYDROLASE
JRNL REF ACTA CRYSTALLOGR.,SECT.D V. 61 920 2005
JRNL REFN ASTM ABCRE6 DK ISSN 0907-4449
REMARK 1
REMARK 2
REMARK 2 RESOLUTION. 1.70 ANGSTROMS.
REMARK 3
REMARK 3 REFINEMENT.
REMARK 3 PROGRAM : CNS 1.0
REMARK 3 AUTHORS : BRUNGER,ADAMS,CLORE,DELANO,GROS,GROSSE-
REMARK 3 : KUNSTLEVE,JIANG,KUSZEWSKI,NILGES, PANNU,
REMARK 3 : READ,RICE,SIMONSON,WARREN
REMARK 3
REMARK 3 REFINEMENT TARGET : ENGH & HUBER
REMARK 3
REMARK 3 DATA USED IN REFINEMENT.
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 1.70
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 28.66
REMARK 3 DATA CUTOFF (SIGMA(F)) : 0.000
REMARK 3 DATA CUTOFF HIGH (ABS(F)) : 1201621.420
REMARK 3 DATA CUTOFF LOW (ABS(F)) : 0.0000
REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 93.7
REMARK 3 NUMBER OF REFLECTIONS : 27967
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT.
REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT
REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM
REMARK 3 R VALUE (WORKING SET) : 0.171
REMARK 3 FREE R VALUE : 0.207
REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.800
REMARK 3 FREE R VALUE TEST SET COUNT : 1350
REMARK 3 ESTIMATED ERROR OF FREE R VALUE : 0.006
REMARK 3
REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN.
REMARK 3 TOTAL NUMBER OF BINS USED : 6
REMARK 3 BIN RESOLUTION RANGE HIGH (A) : 1.70
REMARK 3 BIN RESOLUTION RANGE LOW (A) : 1.81
REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 92.00
REMARK 3 REFLECTIONS IN BIN (WORKING SET) : 4279
REMARK 3 BIN R VALUE (WORKING SET) : 0.1860
REMARK 3 BIN FREE R VALUE : 0.2520
REMARK 3 BIN FREE R VALUE TEST SET SIZE (%) : 4.80
REMARK 3 BIN FREE R VALUE TEST SET COUNT : 215
REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.017
REMARK 3
REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.
REMARK 3 PROTEIN ATOMS : 1776
REMARK 3 NUCLEIC ACID ATOMS : 0
REMARK 3 HETEROGEN ATOMS : 15
REMARK 3 SOLVENT ATOMS : 205
REMARK 3
REMARK 3 B VALUES.
REMARK 3 FROM WILSON PLOT (A**2) : 13.60
REMARK 3 MEAN B VALUE (OVERALL, A**2) : 14.60
REMARK 3 OVERALL ANISOTROPIC B VALUE.
REMARK 3 B11 (A**2) : -1.83000
REMARK 3 B22 (A**2) : 1.42000
REMARK 3 B33 (A**2) : 0.42000
REMARK 3 B12 (A**2) : 0.00000
REMARK 3 B13 (A**2) : 0.00000
REMARK 3 B23 (A**2) : 0.00000
REMARK 3
REMARK 3 ESTIMATED COORDINATE ERROR.
REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.15
REMARK 3 ESD FROM SIGMAA (A) : 0.02
REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00
REMARK 3
REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR.
REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.20
REMARK 3 ESD FROM C-V SIGMAA (A) : 0.12
REMARK 3
REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES.
REMARK 3 BOND LENGTHS (A) : 0.025
REMARK 3 BOND ANGLES (DEGREES) : 2.10
REMARK 3 DIHEDRAL ANGLES (DEGREES) : 24.40
REMARK 3 IMPROPER ANGLES (DEGREES) : 1.53
REMARK 3
REMARK 3 ISOTROPIC THERMAL MODEL : RESTRAINED
REMARK 3
REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA
REMARK 3 MAIN-CHAIN BOND (A**2) : 2.640 ; 1.500
REMARK 3 MAIN-CHAIN ANGLE (A**2) : 3.300 ; 2.000
REMARK 3 SIDE-CHAIN BOND (A**2) : 5.210 ; 2.000
REMARK 3 SIDE-CHAIN ANGLE (A**2) : 6.200 ; 2.500
REMARK 3
REMARK 3 BULK SOLVENT MODELING.
REMARK 3 METHOD USED : FLAT MODEL
REMARK 3 KSOL : 0.45
REMARK 3 BSOL : 55.79
REMARK 3
REMARK 3 NCS MODEL : NULL
REMARK 3
REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHT
REMARK 3 GROUP 1 POSITIONAL (A) : NULL ; NULL
REMARK 3 GROUP 1 B-FACTOR (A**2) : NULL ; NULL
REMARK 3
REMARK 3 PARAMETER FILE 1 : PROTEIN_REP.PARAM
REMARK 3 PARAMETER FILE 2 : GOL_C123S.PARAM
REMARK 3 PARAMETER FILE 3 : WATER_REP.PARAM
REMARK 3 PARAMETER FILE 4 : ION.PARAM
REMARK 3 PARAMETER FILE 5 : NULL
REMARK 3 TOPOLOGY FILE 1 : PROTEIN.TOP
REMARK 3 TOPOLOGY FILE 2 : GOL_C123S.TOP
REMARK 3 TOPOLOGY FILE 3 : WATER.TOP
REMARK 3 TOPOLOGY FILE 4 : ION.TOP
REMARK 3 TOPOLOGY FILE 5 : NULL
REMARK 3
REMARK 3 OTHER REFINEMENT REMARKS: NULL
REMARK 4
REMARK 4 1ZI6 COMPLIES WITH FORMAT V. 2.3, 09-JULY-1998
REMARK 100
REMARK 100 THIS ENTRY HAS BEEN PROCESSED BY PDBJ ON 02-MAY-2005.
REMARK 100 THE RCSB ID CODE IS RCSB032752.
REMARK 200
REMARK 200 EXPERIMENTAL DETAILS
REMARK 200 EXPERIMENT TYPE : X-RAY DIFFRACTION
REMARK 200 DATE OF DATA COLLECTION : 14-MAR-2001
REMARK 200 TEMPERATURE (KELVIN) : 100.0
REMARK 200 PH : 6.50
REMARK 200 NUMBER OF CRYSTALS USED : 1
REMARK 200
REMARK 200 SYNCHROTRON (Y/N) : N
REMARK 200 RADIATION SOURCE : ROTATING ANODE
REMARK 200 BEAMLINE : NULL
REMARK 200 X-RAY GENERATOR MODEL : RIGAKU RU200
REMARK 200 MONOCHROMATIC OR LAUE (M/L) : M
REMARK 200 WAVELENGTH OR RANGE (A) : 1.5418
REMARK 200 MONOCHROMATOR : OSMIC MAXXFLUX CONFOCAL
REMARK 200 OPTICS (GREEN)
REMARK 200 OPTICS : CONFOCAL MIRRORS
REMARK 200
REMARK 200 DETECTOR TYPE : IMAGE PLATE
REMARK 200 DETECTOR MANUFACTURER : RIGAKU RAXIS IIC
REMARK 200 INTENSITY-INTEGRATION SOFTWARE : DENZO
REMARK 200 DATA SCALING SOFTWARE : SCALEPACK
REMARK 200
REMARK 200 NUMBER OF UNIQUE REFLECTIONS : 28238
REMARK 200 RESOLUTION RANGE HIGH (A) : 1.700
REMARK 200 RESOLUTION RANGE LOW (A) : 33.840
REMARK 200 REJECTION CRITERIA (SIGMA(I)) : 0.000
REMARK 200
REMARK 200 OVERALL.
REMARK 200 COMPLETENESS FOR RANGE (%) : 94.4
REMARK 200 DATA REDUNDANCY : 5.350
REMARK 200 R MERGE (I) : 0.09500
REMARK 200 R SYM (I) : NULL
REMARK 200 FOR THE DATA SET : 19.4000
REMARK 200
REMARK 200 IN THE HIGHEST RESOLUTION SHELL.
REMARK 200 HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : 1.70
REMARK 200 HIGHEST RESOLUTION SHELL, RANGE LOW (A) : 1.81
REMARK 200 COMPLETENESS FOR SHELL (%) : 92.0
REMARK 200 DATA REDUNDANCY IN SHELL : NULL
REMARK 200 R MERGE FOR SHELL (I) : 0.22000
REMARK 200 R SYM FOR SHELL (I) : NULL
REMARK 200 FOR SHELL : NULL
REMARK 200
REMARK 200 DIFFRACTION PROTOCOL: SINGLE WAVELENGTH
REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT
REMARK 200 SOFTWARE USED: KNOWN STRUCTURE
REMARK 200 STARTING MODEL: PDB ENTRY 1DIN
REMARK 200
REMARK 200 REMARK: NULL
REMARK 280
REMARK 280 CRYSTAL
REMARK 280 SOLVENT CONTENT, VS (%): 47.56
REMARK 280 MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): 2.36
REMARK 280
REMARK 280 CRYSTALLIZATION CONDITIONS: 0.1M SODIUM CITRATE BUFFER, 1.2M
REMARK 280 AMMONIUM SULFATE, PH 6.5, VAPOR DIFFUSION, HANGING DROP,
REMARK 280 TEMPERATURE 277K
REMARK 290
REMARK 290 CRYSTALLOGRAPHIC SYMMETRY
REMARK 290 SYMMETRY OPERATORS FOR SPACE GROUP: P 21 21 21
REMARK 290
REMARK 290 SYMOP SYMMETRY
REMARK 290 NNNMMM OPERATOR
REMARK 290 1555 X,Y,Z
REMARK 290 2555 1/2-X,-Y,1/2+Z
REMARK 290 3555 -X,1/2+Y,1/2-Z
REMARK 290 4555 1/2+X,1/2-Y,-Z
REMARK 290
REMARK 290 WHERE NNN -> OPERATOR NUMBER
REMARK 290 MMM -> TRANSLATION VECTOR
REMARK 290
REMARK 290 CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS
REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM
REMARK 290 RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY
REMARK 290 RELATED MOLECULES.
REMARK 290 SMTRY1 1 1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY2 1 0.000000 1.000000 0.000000 0.00000
REMARK 290 SMTRY3 1 0.000000 0.000000 1.000000 0.00000
REMARK 290 SMTRY1 2 -1.000000 0.000000 0.000000 24.11300
REMARK 290 SMTRY2 2 0.000000 -1.000000 0.000000 0.00000
REMARK 290 SMTRY3 2 0.000000 0.000000 1.000000 38.44900
REMARK 290 SMTRY1 3 -1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY2 3 0.000000 1.000000 0.000000 35.64150
REMARK 290 SMTRY3 3 0.000000 0.000000 -1.000000 38.44900
REMARK 290 SMTRY1 4 1.000000 0.000000 0.000000 24.11300
REMARK 290 SMTRY2 4 0.000000 -1.000000 0.000000 35.64150
REMARK 290 SMTRY3 4 0.000000 0.000000 -1.000000 0.00000
REMARK 290
REMARK 290 REMARK: NULL
REMARK 300
REMARK 300 BIOMOLECULE: 1
REMARK 300 THIS ENTRY CONTAINS THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT
REMARK 300 WHICH CONSISTS OF 1 CHAIN(S). SEE REMARK 350 FOR
REMARK 300 INFORMATION ON GENERATING THE BIOLOGICAL MOLECULE(S).
REMARK 350
REMARK 350 GENERATING THE BIOMOLECULE
REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN
REMARK 350 BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE
REMARK 350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS
REMARK 350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND
REMARK 350 CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN.
REMARK 350
REMARK 350 BIOMOLECULE: 1
REMARK 350 APPLY THE FOLLOWING TO CHAINS: A
REMARK 350 BIOMT1 1 1.000000 0.000000 0.000000 0.00000
REMARK 350 BIOMT2 1 0.000000 1.000000 0.000000 0.00000
REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000
REMARK 465
REMARK 465 MISSING RESIDUES
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
REMARK 465
REMARK 465 M RES C SSSEQI
REMARK 465 LYS A 234
REMARK 465 LYS A 235
REMARK 465 PRO A 236
REMARK 500
REMARK 500 GEOMETRY AND STEREOCHEMISTRY
REMARK 500 SUBTOPIC: TORSION ANGLES
REMARK 500
REMARK 500 TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS:
REMARK 500 (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN IDENTIFIER;
REMARK 500 SSEQ=SEQUENCE NUMBER; I=INSERTION CODE).
REMARK 500
REMARK 500 STANDARD TABLE:
REMARK 500 FORMAT:(10X,I3,1X,A3,1X,A1,I4,A1,4X,F7.2,3X,F7.2)
REMARK 500
REMARK 500 M RES CSSEQI PSI PHI
REMARK 500 SER A 123 -113.37 60.76
REMARK 900
REMARK 900 RELATED ENTRIES
REMARK 900 RELATED ID: 1DIN RELATED DB: PDB
REMARK 900 THE SAME PROTEIN WITH ONLY C123S MUTATION
REMARK 900 RELATED ID: 1GGV RELATED DB: PDB
REMARK 900 THE SAME PROTEIN WITH ONLY C123S MUTATION AND WITH PMS
REMARK 900 MOITY ON THE RESIDUE 123
REMARK 900 RELATED ID: 1ZI8 RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, E36D, C123S, A134S, S208G, A229V, K234R
REMARK 900 MUTANT AT 1.4 A
REMARK 900 RELATED ID: 1ZI9 RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, E36D, C123S MUTANT AT 1.5 A
REMARK 900 RELATED ID: 1ZIC RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, C123S, R206A MUTANT AT 1.7 A
REMARK 900 RELATED ID: 1ZIX RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, E36D, R105H, C123S, G211D, K234N MUTANT
REMARK 900 AT 1.8 A
REMARK 900 RELATED ID: 1ZIY RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, C123S MUTANT COMPLEXED WITH PMSF AT 1.9 A
REMARK 900 RELATED ID: 1ZJ4 RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, E36D, C123S MUTANT COMPLEXED WITH PMSF AT
REMARK 900 1.7 A
REMARK 900 RELATED ID: 1ZJ5 RELATED DB: PDB
REMARK 900 THE SAME PROTEIN, E36D, C123S, A134S, S208G, A229V, K234R
REMARK 900 MUTANT COMPLEXED WITH PMSF AT 1.7 A
DBREF 1ZI6 A 1 236 SWS P0A114 CLCD_PSEPU 1 236
SEQADV 1ZI6 SER A 123 SWS P0A114 CYS 123 ENGINEERED
SEQADV 1ZI6 ASN A 154 SWS P0A114 LYS 154 CONFLICT
SEQADV 1ZI6 THR A 224 SWS P0A114 ARG 224 CONFLICT
SEQRES 1 A 236 MET LEU THR GLU GLY ILE SER ILE GLN SER TYR ASP GLY
SEQRES 2 A 236 HIS THR PHE GLY ALA LEU VAL GLY SER PRO ALA LYS ALA
SEQRES 3 A 236 PRO ALA PRO VAL ILE VAL ILE ALA GLN GLU ILE PHE GLY
SEQRES 4 A 236 VAL ASN ALA PHE MET ARG GLU THR VAL SER TRP LEU VAL
SEQRES 5 A 236 ASP GLN GLY TYR ALA ALA VAL CYS PRO ASP LEU TYR ALA
SEQRES 6 A 236 ARG GLN ALA PRO GLY THR ALA LEU ASP PRO GLN ASP GLU
SEQRES 7 A 236 ARG GLN ARG GLU GLN ALA TYR LYS LEU TRP GLN ALA PHE
SEQRES 8 A 236 ASP MET GLU ALA GLY VAL GLY ASP LEU GLU ALA ALA ILE
SEQRES 9 A 236 ARG TYR ALA ARG HIS GLN PRO TYR SER ASN GLY LYS VAL
SEQRES 10 A 236 GLY LEU VAL GLY TYR SER LEU GLY GLY ALA LEU ALA PHE
SEQRES 11 A 236 LEU VAL ALA ALA LYS GLY TYR VAL ASP ARG ALA VAL GLY
SEQRES 12 A 236 TYR TYR GLY VAL GLY LEU GLU LYS GLN LEU ASN LYS VAL
SEQRES 13 A 236 PRO GLU VAL LYS HIS PRO ALA LEU PHE HIS MET GLY GLY
SEQRES 14 A 236 GLN ASP HIS PHE VAL PRO ALA PRO SER ARG GLN LEU ILE
SEQRES 15 A 236 THR GLU GLY PHE GLY ALA ASN PRO LEU LEU GLN VAL HIS
SEQRES 16 A 236 TRP TYR GLU GLU ALA GLY HIS SER PHE ALA ARG THR SER
SEQRES 17 A 236 SER SER GLY TYR VAL ALA SER ALA ALA ALA LEU ALA ASN
SEQRES 18 A 236 GLU ARG THR LEU ASP PHE LEU ALA PRO LEU GLN SER LYS
SEQRES 19 A 236 LYS PRO
HET SO4 2719 5
HET SO4 2720 5
HET SO4 2721 5
HET GOL 1101 6
HETNAM SO4 SULFATE ION
HETNAM GOL GLYCEROL
FORMUL 2 SO4 3(O4 S1 2-)
FORMUL 5 GOL C3 H8 O3
FORMUL 6 HOH *199(H2 O1)
HELIX 1 1 ASN A 41 GLN A 54 1 14
HELIX 2 2 LEU A 63 GLN A 67 5 5
HELIX 3 3 ASP A 77 PHE A 91 1 15
HELIX 4 4 ASP A 92 HIS A 109 1 18
HELIX 5 5 SER A 123 GLY A 136 1 14
HELIX 6 6 GLY A 148 VAL A 159 5 12
HELIX 7 7 PRO A 175 GLY A 187 1 13
HELIX 8 8 VAL A 213 ALA A 229 1 17
HELIX 9 9 PRO A 230 GLN A 232 5 3
SHEET 1 A 2 ILE A 8 GLN A 9 0
SHEET 2 A 2 THR A 15 PHE A 16 -1 O PHE A 16 N ILE A 8
SHEET 1 B 7 ALA A 18 GLY A 21 0
SHEET 2 B 7 ALA A 57 PRO A 61 -1 O CYS A 60 N LEU A 19
SHEET 3 B 7 ALA A 28 ALA A 34 1 N ILE A 31 O ALA A 57
SHEET 4 B 7 SER A 113 TYR A 122 1 O GLY A 118 N VAL A 32
SHEET 5 B 7 ARG A 140 TYR A 144 1 O VAL A 142 N LEU A 119
SHEET 6 B 7 ALA A 163 GLY A 168 1 O LEU A 164 N GLY A 143
SHEET 7 B 7 LEU A 192 TYR A 197 1 O HIS A 195 N MET A 167
CISPEP 1 ALA A 26 PRO A 27 0 -0.16
CRYST1 48.226 71.283 76.898 90.00 90.00 90.00 P 21 21 21 4
ORIGX1 1.000000 0.000000 0.000000 0.00000
ORIGX2 0.000000 1.000000 0.000000 0.00000
ORIGX3 0.000000 0.000000 1.000000 0.00000
SCALE1 0.020736 0.000000 0.000000 0.00000
SCALE2 0.000000 0.014029 0.000000 0.00000
SCALE3 0.000000 0.000000 0.013004 0.00000
TER 1777 SER A 233
MASTER 265 0 4 9 9 0 0 6 1996 1 21 19
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