W250 Nucleic Acids Research, 2008, Vol. 36, Web Server issue Figure 6. PX domain proteins have strong similarities at the N- and C-termini, but not in the loop region indicated by the arrow. PX domain pFlexAna is particularly useful for proteins exhibiting domain moments, but applying it to proteins with more subtle conformational changes and without major domain movements can also be informative. In particular, we set parameter k = 1 when we expect only a single cluster corresponding to the aligned protein core. For example, Figure 6 compares two PX domain proteins [1KMD (19) and 1GD5 (20)]. The N- and C-termini of the two proteins are conserved, but the loop region in between contains several insertions/deletions. Most structural alignment methods attempt to align these two proteins by super- imposing their entire backbones globally. While this may be useful for some purposes, single global alignments may be skewed as they include the loop region. A more informative view of the evolutionary impact on conforma- tional structure is shown by aligning only the conserved segments. On the PX domains, pFlexAna automatically ﬁnds the fragments of tightest similarity, distinct from the loop region, and it presents the clustered alignment result without skew from the inserted amino acids. DISCUSSION pFlexAna is a new tool for protein structure comparison. It detects and displays conformational changes in remotely related proteins, without relying on sequence homology. Our tests show that pFlexAna helps in anal- yzing a wide variety of conformational changes. It can detect conformational changes in proteins with identical sequences or those with very low sequence similarity. It can detect dramatic domain movements as well as smaller, more subtle conformational changes. The output of pFlexAna helps the analysis of conforma- tional changes in several ways. pFlexAna uses a reliable statistical test of structural similarity to demarcate the protein regions that undergo conformational changes. It also clusters the protein fragments that move in tandem. The clustering helps in ﬁnding domains comprised of frag- ments that are discontiguous and out of order along the sequences, e.g. in the case of the maltose-binding protein (Figure 3). Visualization of such information, provided in pFlexAna’s output, helps the user to scan for hinge locations, active binding sites, domain movements, etc. For more detailed analysis, the user may use the quantitative information provided in pFlexAna’s output, e.g. use the provided transformation matrix for alignment to compute the angle of bending at a particular hinge. pFlexAna’s output may also help users interested in predicting conformational motion. They may use the information that pFlexAna provides as a starting point and combine it with other methods such as targeted molecular dynamics (21), where an MD simulation trajectory is ‘directed’ from an inital conformation towards an alternative target conformation. pFlexAna uses purely structural information to match two proteins. While this is often advantageous as it does not rely on sequence homology, it can potentially cause false positive matching. For example, for proteins with many secondary structure elements (SSEs), it is possible for unrelated long alpha helices or other common motifs comprised of multiple SSEs to be identiﬁed as aligned fragment pairs. Comparing glutamine amidotransferase (1MOQ) with a ubiquitin ligase (1ND7) matches two pairs of highly similar a-helices, with RMSD values of 0.22 A˚ and 1.64 A˚ , respectively. While such aligned pairs are justiﬁed based on purely structural similarity, they may not be biologically signiﬁcant to form a basis for inferring conformational changes. In the future, we intend to ﬁlter out such false positives by incorporating secondary structure or sequence information during the matching. ACKNOWLEDGEMENTS I.M. and C.V. gratefully acknowledge support by Biomedical Research Council of A*STAR, Singapore. L.T.K. was supported by a Lee Kuan Yew Postdoctoral Fellowship. Funding to pay the Open Access publication charges for this article was provided by the NUS AcRF grant R-252-000-342-112. Conﬂict of interest statement. None declared. REFERENCES 1. Swain,J.F. and Gierasch,L.M. (2006) The changing landscape of protein allostery. Curr. Opin. Struct. Biol., 16, 102–108. 2. Huang,L., Kinnucan,E., Wang,G., Beaudenon,S., Howley,P.M., Huibregtse,J.M. and Pavletich,N.P. (1999) Structure of an e6ap- ubch7 complex: insights into ubiquitination by the e2-e3 enzyme cascade. 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