After starting DomainFinder, load your protein structure using ``Load reference structure...'' in the File menu. DomainFinder will then propose reasonable values for the number of calculated modes and the number of modes kept for analysis. However, you may modify these numbers if you wish. The number of calculated modes determines the accuracy of the normal mode calculations; the more modes you calculate, the better the description of the motions. The value proposed by DomainFinder is a rather small, but reasonable, value; you might want to increase it to obtain better results. In contrast, the number of modes kept for analysis has no influence on the quality of the results. It exists purely for efficiency reasons: keeping all calculated modes would slow down the analysis and waste disk space if the modes are saved in a file. There is little reason to increase the proposed number, because the additional modes typically do not describe domain motions are are therefore not useful for the domain analysis.
To start the normal mode calculation, press the button labeled ``Calculate modes''. Depending on the size of the protein and the speed of your computer, the calculation can take from a few seconds to a few hours. For example, the calculation of 100 modes for a 500-residue protein takes 90 seconds on a 90 MHz Pentium PC; the same calculation takes 22 seconds on an IBM RS/6000 Model 43P-140. When the calculation is finished, DomainFinder displays a list of the modes with non-zero frequencies (the first number is seven, because there are six zero-frequency modes) and the actual number of calculated modes. This number is in general different from the one you entered, because not all values are possible for technical reasons (see the discussion on Fourier bases in Ref. 2). If the number of modes you ask for is equal to or larger than the number of residues in the protein, DomainFinder will calculate all possible modes (three times the number of residues), because calculating fewer modes would not be more efficient.
You must now select the modes that you want to use for the deformation and domain analysis. To help you with this choice, the normal mode list indicates the average deformation energy per residue for each mode. As explained in section 2, a deformation energy is associated with every atom; low values characterize rigid regions, whereas high values indicate flexible regions. A low average deformation energy thus indicates a mode with large rigid regions, which has a good chance of describing domain motions. There is no simple recipe for selecting an optimal set of modes (otherwise DomainFinder would apply it automatically!). As a general guideline, look for jumps in the average deformation energy from one mode to the next, and choose all modes before such a jump; there is no justification for selecting only some out of a set of modes with very similar deformation energies. Start with few modes, and add more modes only if you are not satisfied with the amount of detail in the domain analysis.
After selecting the modes, you must choose the deformation threshold that defines which regions are sufficiently rigid to be candidates for domains. This choice is related to the mode selection; to have a reasonable number of rigid regions, the deformation threshold should be of the same order of magnitude as the average deformation energy of the modes you have chosen. A higher deformation threshold leads to larger rigid regions; if you find later that your domains cover too small a part of the protein, you should increase the deformation threshold.
The following steps are the same as for an analysis based on comparing two conformations, and are described in sections 6 and 7.
If you wish to stop working on an analysis and continue it later, you can save time by saving the already calculated normal modes to a file and then later loading this file instead of the input structure. This is done using the File menu entries ``Save modes...'' and ``Load modes...''.