| SIMULATED ANNEALING, RESTRAINED MOLECULAR DYNAMICS | DESCRIPTION OF THE STRATEGY USED FOR NMR STRUCTURE CALCULATION AND REFINEMENT: NOE CROSS-PEAKS WERE DIVIDED INTO THREE CATEGORIES AND ASSIGNED DISTANCE RANGES ACCORDING TO THEIR INTENSITY: STRONG (<0.29 NM); MEDIUM (<0.42 NM); WEAK (<0.57 NM). PEAK INTENSITIES WERE ESTIMATED FROM THE NUMBER OF CONTOURS IN NOESY SPECTRUM. 23 3JHNA COUPLING CONSTANTS WERE EXTRACTED FROM DQF-COSY SPECTRUM, AND CONVERTED TO PHI-ANGLES ACCORDING THE KARPLUS EQUATION. THE STRUCTURE CALCULATIONS USED MODIFIED AB INITIO SIMULATED ANNEALING (SA. INP) AND REFINEMENT (REFINE. INP) PROTOCOLS FROM THE X-PLOR PROGRAM PACKAGE WHICH INCLUDED FLOATING ASSIGNMENT OF PROCHIRAL GROUPS, REDUCED PRESENTATION FOR NON-BOND INTERACTIONS FOR PART OF THE CALCULATION, REFINEMENT AGAINST CONFORMATIONAL DATABASE AND DIRECT REFINEMENT AGAINST 3JHNA COUPLING CONSTANTS. IN EACH ROUND OF STRUCTURE CALCULATION, 100 STRUCTURES WERE CALCULATED FROM TEMPLATES WITH RANDOM BACKBONE TORSION ANGLES. IN THE CONFORMATIONAL SEARCH PHASE 60 PS OF MOLECULAR DYNAMICS WERE SIMULATED AT 2000 K. THE REFINEMENT COMPRISED A TWO-PHASE COOLING PROCEDURE TREATING THE NON-BONDED INTERACTIONS BETWEEN ALL ATOMS BY A REPULSIVE ('REPEL') POTENTIAL. IN THE FIRST REFINEMENT STAGE, THE SYSTEM WAS COOLED FROM 2000 K TO 1000 K WITHIN 135 PS, CONCOMITANTLY INCREASING THE FORCE CONSTANTS TO THEIR FINAL VALUES. IN THE NEXT STAGE OF THE CALCULATION THE SYSTEM WAS COOLED FROM 1000 K TO 100 K WITHIN 90 PS, APPLYING THE HIGH FORCE CONSTANTS OBTAINED AT THE END OF THE PREVIOUS COOLING STAGE. DURING ALL STAGES OF THE CALCULATION A TIMESTEP OF 3 FS WAS USED. THEN, 500 STEPS OF POWELL ENERGY MINIMIZATION WERE PERFORMED, USING AN ATTRACTIVE LENARD-JONES POTENTIAL, BUT NO EXPLICIT ELECTROSTATICS AND RAMACHANDRAN DATABASIS POTENTIAL. OF THE 240 RESULTING STRUCTURES, THOSE 10 STRUCTURES THAT SHOWED THE LOWEST ENERGY AND THE LEAST VIOLATION OF THE EXPERIMENTAL DATA WERE SELECTED FOR FURTHER CHARACTERIZATION. GEOMETRY OF THE STRUCTURES AND ELEMENTS OF SECONDARY STRUCTURE WERE ANALYZED USING PROCHECK AND DSSP. | X-PLOR |
