Nucleic Acids Research, 2008, Vol. 36, Web Server issue W247 Figure 1. Superposition of the structures of ubiquitin ligases WWWP1 and E6AP. The two diﬀerent superpositions, produced automatically by pFlexAna, are obtained by aligning a pair of fragments (marked in red and green) with high structural similarity. Dotted ovals annotate the parts of the proteins that are well aligned in the superpositions. To see the magnitude of the conformational change, note that the pose of E6AP (blue) remains the same in both (a) and (b). Only the pose of WWWP1 (orange) changes. Figure 2. Superposition of the bound (red) and unbound (blue) structures of the maltose-binding protein. (a) FATCAT aligns the entire protein structures. (b) pFlexAna correctly identiﬁes two rigid domains that undergo hinge bending. The proteins are shown aligned on one domain. and usually it can only explore conformational changes that are small in magnitude. An alternative approach is to compare directly the structures of a protein in diﬀerent conformations. There are several good methods for this (4,6–8), but they can be applied only if the structures of the same protein in diﬀerent conformations are available. It is thus sometimes necessary to compare conforma- tions of related, but diﬀerent proteins in order to infer conformational changes. One possibility is to perform sequence alignment as a preprocessing step to match the proteins and then detect conformational changes between them (9). This method, however, is restricted to proteins with 90% sequence identity (9). As a complementary approach, for cases with low sequence homology, one may attempt structural align- ment i.e. matching the proteins based on structural simil- arity instead of sequence similarity. However, both rigid and ﬂexible structural alignment methods focus on ﬁnding a single best ﬁt between the protein structures, and may ignore small yet signiﬁcant conformational changes. Consider, for example, the relatively simple case of a same protein in two conformations. Given the bound and unbound structures [PDB codes 3MBP (10) and 1LLS:A (11)] of the maltose-binding protein, two popular struc- tural alignment methods, CE (12) and FATCAT (13), align the entire structures together without recognizing the hinge-like conformational change occurring between the two domains of the protein. FlexProt (14), on the other hand, requires the user to know the number of hinges in order to interpret its results. What we need in this case is a method that automatically detects the ‘discontinuity’ in structural similarity at the hinge due to the conforma- tional change and matches the two domains on each side of the hinge separately. See Figure 2 for a comparison between the results from FATCAT and pFlexAna. In general, pFlexAna aims to detect such conformational changes for two related, but diﬀerent proteins. Further- more, it tries to identify and group together those protein fragments that move in tandem. METHODS Input The pFlexAna server takes as input two protein structures in PDB format, which may be uploaded or speciﬁed using PDB codes. The user may also specify chain identiﬁers or a restricted range of residues for each ﬁle. Finally, there are two optional parameters that the user may adjust. The parameter k is the desired number of clusters for grouping