Results and analyses

Two types of analysis are performed during an ARIA run. The NMR restraints are analyzed at each iteration, and the obtained conformers are analyzed at the end of the last in vacuo iteration and at the end of the water refinement step. The restraint and conformer analyses are described below.

Analysis of the NMR restraints

They are performed at each iteration by CNS, because the sorting of the NMR restraints are based on statistics on the restraints violations. In ARIA, each restraint is the sum of contributions, each contribution beging applied between a pair of nuclei or equivalent nuclei.

At each iteration, the violations and the violation RMS of distance restraints are analyzed, and the most often violated contributions are discarded (see the page Project  to learn more about how to control this process), and new lists of ambiguous and unambiguous distance restraints are produced. During the analysis of the restraints, spectrum peaks having the same contributions are merged. The list of merged peks is an output file of the iteration.

A report file is produced, as well as several files noe_restraints.*, which have the following content:

noe_restraints.ambig	list of ambiguous peaks
noe_restraints.assignments	list of all assignments
noe_restraints.merged	list of merged peaks
noe_restraints.unambig	list of unambiguous peaks
noe_restraints.violations	list of the distance restraint violations

Each of these file has a header containing more precise information about their content. All files are located into the itxxx directory, where xxx is the iteration number.

The report file contains general statistics about the restraints, given for the whole set of restraints as well as for each analyzed spectrum. In particular, for each spectrum, the histogram of the contribution to each peak is given. An example of report file can be found here.

The floating chirality assignment is used to assign the chemical shifts of the two substituents of a prochiral center stereospecifically. In proteins, these are the two methylene protons or the methyl protons of the isopropyl groups of valine or leucine. A resonance matching one of the chemical shifts in the proton dimensions potentially involves either of the two prochiral substituents. ARIA compensates this lack of information by testing both alternatives during structure calculation. For each calculated conformer conf.pdb, the energetically preferred options are written to a file conf.float, which is located in the corresponding structure/itxxx directory.

Analysis of the conformers by CNS

An analysis of the conformer geometry and of the restraint violation is performed by CNS for the last iteration in vacuo, on the best-energy conformers. The number of selected conformers is an input of the project. The results files are stored in the directory analysis of the last iteration, and the CNS output files are stored in the directory analysis/cns.

The .viol files contain the analysis of the violation per restraint for each conformer analyzed. The .pdb files are the conformers superimposed to minimize the RMSD, and the ave.pdb file is the mean conformattion.

The .disp files contain the analysis results:

cop.disp	circular order parameter
couplings.disp	violation statistics on J coupling restraints
dihedrals.disp	violation statistics on dihedral restraints
energies.disp	empirical energies
geom.disp	geometry analysis
noe.disp	violation statistics on NOE restraints
enerms.disp	ensemble RMSD as a function of the number of structures sorted in increasing (total or restraint) energies. calculated on all conformers of the iteration.
rmsd.disp	ewo-by-two RMSD on all conformers of the iteration.
print_dist_all.disp	violated distance restraints
print_dist_hbond.disp	violated hbond distance restraints on each selected conformer.
print_noe_all.disp	violation statistics on NOE distances
print_noe_ambig.disp	violation statistics on ambiguous NOE restraints
print_noe.disp	violated NOE distances
print_noe_unambig.disp	violation statistics on unambiguous NOE restraints
ramachandran.disp	ramachandran plot for each selected conformer
rmsave.disp	coordinate RMSD value between selected conformers
rmsdseq.disp	sequential RMS differences between coordinates
sani.disp	violation statistics on residual dipolar couplings

Analysis of the conformers quality

For the last iteration, and for the refinement step in water, in addition to the restraint analysis, quality analyses of the conformers are performed. According to the specifications in the project file, quality analyses can be performed using PROCHECK, WHATIF or PROSAII. The files obtained with the programs are processed in order to obtain synthetic reports:

quality_checks	Summarization of all quality analyses
quality_checks.tex	Summarization of all quality analyses: latex file
quality_checks.procheck	Concatenation of the files .sum obtained for each analyzed conformer
quality_checks.whatif	Deviation of the WHATIF parameters from their standard values encountered in crystallographic structures
quality_checks.whatif_fulchk	Full WHATIF report