research papers
Secondarystructure matching (SSM), a new tool for fast protein structure alignment in three dimensions
^{a}European Bioinformatics Institute, Genome Campus, Hinxton, Cambridge CB10 1SD, England
^{*}Correspondence email: keb@ebi.ac.uk
The present paper describes the SSM algorithm of protein structure comparison in three dimensions, which includes an original procedure of matching graphs built on the protein's secondarystructure elements, followed by an iterative threedimensional alignment of protein backbone C atoms. The SSM results are compared with those obtained from other protein comparison servers, and the advantages and disadvantages of different scores that are used for structure recognition are discussed. A new score, balancing the r.m.s.d. and alignment length , is proposed. It is found that different servers agree reasonably well on the new score, while showing considerable differences in r.m.s.d. and .
1. Introduction
Protein function is in significant part determined by spatial structure. It is commonly believed that the threedimensional fold has a major impact on the ability of a protein to bind other proteins or ligands (drugs), stability and purely mechanical aspects of protein behaviour. The similarity analysis of protein structure is therefore a vital step in understanding that protein's role in the machinery of life. Comparison of protein structures is also essential for estimating the evolutionary distances between proteins and protein families.
Currently, there are more than 28 000 structures in the Protein Data Bank (PDB) (Berman et al., 2000), 10–20 structures being deposited on a daily basis. Analysis of the evergrowing number of known structures requires efficient tools for protein structure alignment in three dimensions. In contrast to commonly known sequence alignment (Smith & Waterman, 1981), threedimensional alignment is based on the comparison of geometrical positions, rather than biochemical properties, of aminoacid residues. Two residues are considered as aligned if they satisfy certain distance and orientation criteria at the best mutual superposition of the structures. While high sequence similarity almost always implies structural similarity, the opposite is not true. It is therefore expected that threedimensional alignment will provide more significant clues to protein function and properties than sequence alignment alone.
Several approaches to protein structure alignment have been explored over the past decade. The techniques used include comparison of distance matrices (DALI; Holm & Sander, 1993), analysis of differences in vector distance plots (Orengo & Taylor, 1996), minimization of the soapbubble surface area between two protein backbones (Falicov & Cohen, 1996), dynamic programming on pairwise distances between the proteins' residues (Subbiah et al., 1993; Gerstein & Levitt, 1996, 1998) and secondarystructure elements (SSEs) (Singh & Brutlag, 1997), threedimensional clustering (Vriend & Sander, 1991; Mizuguchi & Go, 1995), graph theory (Mitchell et al., 1990; Alexandrov, 1996; Grindley et al., 1993), combinatorial extension of alignment path (CE; Shindyalov & Bourne, 1998), vector alignment of SSEs (VAST; Gibrat et al., 1996), depthfirst recursive search on SSE (DEJAVU; Kleywegt & Jones, 1997) and many others (Zuker & Somorjai, 1989; Taylor & Orengo, 1989; Godzik & Skolnick, 1994; Russell & Barton, 1992; Sali & Blundell, 1990; Barakat & Dean, 1991; Leluk et al., 2003; Jung & Lee, 2000; Kato & Takahashi, 2001).
None of the existing methods gives an exact solution to the problem. Typically, all the methods agree relatively well on the alignment of highly similar structures but often disagree over details if the similarity is low. Partially, this discrepancy is due to the absence of a commonly accepted measure for structural similarity. Most similarity measures are based on the evaluation of the size of common substructures, for example the length of alignment (the longer, the better), and a measure of the distance between them, such as r.m.s.d. (the lower, the better). However, except for the case of highly similar structures, it is always possible to enlarge the common substructures at the expense of the distance measure between them. Therefore, algorithms of threedimensional alignment typically involve a number of heuristic elements and empirical parameters, which naturally causes differences in results. The effect of employed heuristics or choice of empirical parameters are rarely, if ever, published as systematic studies. There are few data on the comparison of threedimensional alignments produced by different algorithms.
Structural alignment of proteins is known to be a computationally expensive procedure. Alignment of a new structure of a few hundred residues to the whole of the PDB with publicly available web servers (DALI, VAST, CE, DEJAVU and some others) may take several hours, with response time growing sharply as the size of the query structure increases. Our aim was to provide the community with an interactive web server, which would be capable of delivering protein structure alignments and database searches in less than a minute, with high quality of alignments. The goal was achieved with the help of an advanced graphmatching algorithm, recently developed for serving structural queries in the EBIMSD database (Krissinel & Henrick, 2004). The new tool, SSM, has been available for public use from June 2002 at https://www.ebi.ac.uk/msdsrv/ssm . The efficiency of the structurealignment algorithm, described below in this paper, was found sufficient for serving all queries in real time, and therefore, in contrast to most of the other similar servers, SSM does not maintain a database of prealigned structures. Furthermore in this paper, we compare SSM with some publicly available web servers in order to examine the quality of alignments and to demonstrate the range of difference between the servers. Finally, we discuss the problem of measuring the quality of threedimensional alignments for more reliable identification of potentially significant matches.
2. Graphtheoretical approach to matching protein structures
Problems of structure comparison and recognition are conveniently addressed by the graphtheoretical approach (see e.g. Rouvray et al., 1979, and references therein). The approach typically includes three major steps: (a) graph representation of the objects in question, (b) matching the graphs representing the objects and (c) evaluating the common subgraphs found in order to form conclusions about similarity.
Traditionally, threedimensional graphs of chemical structures connect all atoms with distancelabelled edges (see e.g. Gardiner et al., 2000) and should have special labels for atoms representing chiral centres in order to distinguish between mirrorreflected structures. The graphs are then matched with a tolerance to the difference in edge lengths using one of many algorithms available [see the review by Raymond & Willett (2002)]. This approach, however, is not applicable to protein structures because of the prohibitively high cost of graph matching. One of the most frequently used optimal graphmatching algorithms, based on maximal clique detection (Levi, 1972; Bron & Kerbosch, 1973), has time complexity of the order of O[(mn)^{n}], where n and m () denote the size of input graphs. This limits the application of this algorithm to graphs having 20–30 unlabelled vertices. Nonoptimal algorithms give an approximation to the optimal (exact) solution at a lower cost; however, the quality of approximation is not well controllable. The fastest optimal algorithm, based on the decisiontree approach, has been reported by Shearer et al. (2001). This algorithm shows time complexity of only O(2^{n}n^{3}). However, the algorithm is not applicable to the matching of threedimensional graphs because of its space complexity, depending exponentially on the number of vertex and/or edge labels involved (edge labels of threedimensional graphs are derived from the edge length and thus form a nearly continuous label space). Recently we have described a new optimal bactracking algorithm for common subgraph isomorphism (Krissinel & Henrick, 2004), CSIA, which represents an advancement of the widely known algorithm of Ullman (1976) for exact subgraph isomorphism. The time complexity of CSIA is bounded by O(m^{n+1}n), which makes it applicable to graphs having up to unlabelled vertices. It follows from the above that even in the case of a simplified representation of proteins by their backbone C atoms, the traditional approach can be applied only to the shortest protein chains.
Size limitations of the graphtheoretical approach may be overcome if less elementary objects are used as graph vertices (Bessonov, 1985; Raymond et al., 2002). Thus, protein secondarystructure elements represent a natural and convenient set of objects for building threedimensional graphs, partly because secondary structure provides most of the protein functionality and is often conserved through the evolution of the molecule. The idea of using SSEs as elementary motifs for the identification of protein folds was exploited in many studies (Mitchell et al., 1990; Grindley et al., 1993; Gibrat et al., 1996; Singh & Brutlag, 1997; Kleywegt & Jones, 1997). The largest proteins contain up to 100 SSEs per chain, which form a very big graph for optimal graphmatching algorithms. However, as we shall see, using SSEs as graph vertices results in a variety of vertex and edge labels, which considerably speeds up most graphmatching algorithms, including CSIA. We calculated SSEs with the help of the algorithm PROMOTIF (Hutchinson & Thornton, 1996).
Most details of protein fold may be expressed in terms of just two types of SSEs, namely helices and strands. SSEs may be used as graph vertices, v_{i}, with composite labels {T_{i},L_{i}}, where T_{i} denotes the type of vertex (helix or strand, and if it is a helix then what type of helix) and L_{i} specifies the number of residues in the ith SSE. Any two vertices, v_{i} and v_{j}, in the graph are connected by an edge e_{ij}. Edge labels are composed so as to describe the geometry of mutual position and orientation of the connected SSEs, as shown in Fig. 1.
The SSEs are represented by the vectors where
for helices and
for strands (Singh & Brutlag, 1997). In (1) and (2), indices p and q denote the serial numbers of the first and last residues in the SSE, and we neglect strands shorter than three residues and helices shorter than five residues. Each edge, e_{ij}, is then labelled with a property vector C_{i},C_{j}}, where is the edge length (in Å), is the angle between the edge and vertices v_{i}/v_{j}, and is the torsion angle between v_{i} and v_{j}. N_{i/j} is the serial number of v_{i}/v_{j} in their protein chains (as counted from N to C termini), C_{i/j} is the vertex chain identifier. Both N_{i/j} and C_{i/j} are used for controlling the SSE connectivity along the chains (see below).
The set of vertices, edges and their labels gives a full definition of a graph. In order to compare (match) the graphs, a graphmatching algorithm should also be provided with a set of rules for the comparison of individual vertices and edges. Obviously, these rules may be formulated in a number of different ways, each of which would involve a number of empirical parameters to be chosen in the course of multiple trials. In our definition, vertices v_{i} and v_{j} compare if
Two edges, e_{ij} and e_{kl}, are considered as comparable if all of the following hold true:
The tolerances , , , , , and , used in (3)–(7), are chosen empirically. In our implementation, they are tabulated for five levels of match specificity, as listed in Table 1. Parameters for the `Normal' level were defined so as to maximize the number of correct fold identifications at crossmatching the SCOP (Murzin et al., 1995) domains. Levels of higher and lower specificity were chosen more arbitrarily, in order to provide a facility for widening or narrowing the search when necessary.

As seen from expressions (3) and (4), we use both relative and absolute differences in vertex and edge lengths for the comparison (thus allowing for larger absolute differences for longer vertices and edges), while only absolute differences in angles [cf. equation (5)] are analysed.
Comparison of torsion angles allows one to distinguish between mirrorsymmetry mates. Apparently, this differentiation was not made by Singh & Brutlag (1997) and Mitchell et al. (1990), although it was taken into account in other studies (Mizuguchi & Go, 1995; Grindley et al., 1993). It should be realized, however, that if edge–vertex angles are small even a slight difference between them in the compared structures may cause a significant disagreement in torsion angles. We therefore compare only signs of torsion angles, which is sufficient for distinguishing between the symmetry mates, and only if both vertex vectors and the edge are far from collinearity [cf. equation (6)].
Until now we considered threedimensional arrangements of SSEs regardless of their ordering along the protein chain. Usually the connectivity of SSEs is significant; however, there are situations where it may or should be neglected (e.g. comparison of mutated or engineered proteins, or geometry of active sites). In previous studies, the SSE connectivity was either preserved (Singh & Brutlag, 1997) or, apparently, neglected (Mitchell et al., 1990; Grindley et al., 1993; Mizuguchi & Go, 1995). In order to handle the connectivity in a more flexible way, we have introduced a special function, Connect (e_{ij},e_{kl}) [cf. equation (7)], providing for the following three options:
(i) Connectivity of SSEs is neglected. Connect (e_{ij},e_{kl}) always returns true. Motifs A and B, shown in Fig. 2, would then match fully as .
(ii) `Soft' connectivity. The general order of matched SSEs along their protein chains is the same in both structures, but any number of missing or unmatched SSEs between the matched ones is allowed. In this case, Connect (e_{ij},e_{kl}) returns false if
Matching motifs A and B from Fig. 2 then yields five maximal common submotifs of size 2: , , , and .
(iii) `Strict' connectivity. Matched SSEs follow the same order along their protein chains and may be separated only by an equal number of matched or unmatched SSEs in both structures. Connect (e_{ij},e_{kl}) returns false if
Matching motifs A and B from Fig. 2 then yields the only maximal common submotif of size 2: .
Matching threedimensional graphs built on secondarystructure elements gives a correspondence between groups of residues of the compared proteins, which allows for preliminary identification of protein folds and rough estimation of structural similarity. Fine comparative analysis requires information on the correspondence between individual residues, including those not found in SSEs. In order to obtain threedimensional alignment of individual residues, we represent them by their atoms and apply an additional procedure of aligning the latter in three dimensions, using the results of graph matching as a starting point. The alignment procedure is described in the next section.
3. C_{α} alignment in three dimensions
Alignment problems are traditionally approached by the technique of dynamic programming (Smith & Waterman, 1981), which may also be applied to structure alignment (Subbiah et al., 1993; Gerstein & Levitt, 1996 1998; Singh & Brutlag, 1997). This technique, however, is not applicable if SSE connectivity is neglected and best alignment is achieved at misconnected SSEs (cf. the discussion above). We therefore employ a different procedure, which optimizes a quality function calculated at best superposition of aligned structures. The procedure is generally similar to those used in other studies (see e.g. Singh & Brutlag, 1997; Kleywegt & Jones, 1997); however, it involves a number of empirical elements, which are introduced and adjusted in the course of analysis of thousands of alignments. We therefore describe our algorithm `as is', without discussion of its differences from similar techniques and exhaustive justification.
Our procedure is based on fast optimal superposition (FOS) of two sets of points in threedimensional space (in our case, the positions of the C atoms of the two structures to be aligned), provided that correspondence between them is known. Several FOS techniques are available (McLachlan, 1972; Kabsch, 1976, 1978). We used a singular value decomposition of the correlation matrix, following the method described by Lesk (1986). The rotoinversion, if detected, is eliminated by changing the sign of the singular vector corresponding to the minimal singular value. The procedure is described in Appendix A.
Once the structures are superposed, their C atoms may be mapped onto each other using the procedure described below. The initial superposition of the structures is obtained by applying FOS to the representing vectors of matched SSEs [equations (1) and (2)]. This approach, however, does not work well if the SSE vectors , forming a common SSE subgraph, are collinear. Applied to a set of collinear vectors, FOS yields a rotation matrix with arbitrary rotation about the vectors. We therefore add the edgerepresenting vectors to the sets of matched if the minimal absolute value of the cosine between any two of them exceeds 0.8. If addition of edgerepresenting vectors does not decrease the minimal cosine or if, in the case of structures with low similarity, the maximal common SSE subgraph includes only one SSE, we explore the whole rotation about the ambiguous axis in order to achieve a maximal overlap of other (type and directioncompatible) SSEs or individual C atoms.
The mutual correspondence (mapping) between C atoms of superposed structures is found through analysis of the distances between them. While the simplest contactseeking approach would suffice in many cases, we found that for the best quality of alignment one should employ a more sophisticated technique. We suggest that the following steps are performed in order of their numbering.
(i) Mapping C atoms of matched SSEs. For each pair of matched SSEs, we find n_{a} (n_{a} = 3 for strands and n_{a} = 4 for helices) neighbouring pairs of C atoms with minimal separation, mark them as mapped and then expand the mapping to the ends of the SSEs, leaving no unmapped atoms between the mapped ones (see Fig. 3). The value of n_{a} = 4 for helices ensures that full helix turns are always mapped properly, even if there is only a partial overlap between the helices.
(ii) Mapping C atoms of nonmatched SSEs. All pairs of nonmatched SSEs. v^{1}_{i} and v^{2}_{j}, which are of the same type and collinear with cosine greater than 0.7, are ranged in order of increasing r.m.s.d. of their closest n_{a} C atoms (dark atoms in Fig. 3), and only pairs with the lowest r.m.s.d. () are left in the list. If the r.m.s.d. of the two pairs (v^{1}_{i},v^{2}_{j}) and (v^{1}_{i},v^{2}_{k}) is less than R_{c}, only one pair with the lowest r.m.s.d. is left (the superscripts stand for structure ID). Then the C atoms of all SSE pairs in the list are mapped as described above, starting from the pair with the lowest r.m.s.d. Before mapping an SSE pair, it is necessary to check that the mapping will not violate the connectivity of already mapped atoms (as explained in Fig. 4), if connectivity should be preserved (cf. §2). The preliminary ranging of SSE pairs on increasing r.m.s.d. ensures that only the bestoverlapping SSEs will be mapped in the case of a connectivity conflict.
(iii) Expansion of contacts. If atom A of structure 1 and atom B of structure 2 form a contact, the distance between A and B is less than the distance between A and any atom of chain 2, except B, and less then distance between B and any atom of chain 1, except A. Finding contacts is an expensive procedure, unless a bricking algorithm is employed [see for example the program CONTACT by Tadeusz Skarzynski in the CCP4 suite (Collaborative Computational Project, Number 4, 1994)]. Contacts are calculated for all yet unmapped but mappable pairs of atoms and are ranged by increasing contact distance, and only contacts with contact distances shorter than R_{c} are left in the list. We consider a pair of atoms as unmappable if one atom belongs to a helix (unless closer than three residues to the helix ends) and another one belongs to a nonhelical part of the protein chain. Starting with the shortest contact, contacting C atoms are mapped onto each other, provided that such mappings do not violate the chain connectivity (cf. Fig. 4). After consideration of all contacts, the procedure tries to map all remained mappable pairs of atoms, starting from pairs that adjoin the contacts, as shown in Fig. 5.
(iv) Quality filter. The previous steps result in the mapping of up to min(N_{1},N_{2}) C atoms, where N_{1} and N_{2} are the number of residues in the aligned structures. In general, such mapping includes both similar and less similar substructures. Quite often, the quality of alignment may be improved by unmapping C atoms of less similar parts. Usually this is achieved by introducing a cutoff distance of about 2–4 Å. Such an approach, however, does not work well in many instances where one structure is a distorted (by a few Å) replica of another, and therefore the r.m.s.d. is not a good measure of the alignment quality. An intuitive understanding of structural similarity suggests contradictory requirements of achieving a lower r.m.s.d. and a higher number of mapped (aligned) residues . This contradiction may be eliminated, in the first approximation, by a score that represents a ratio of and the r.m.s.d. We therefore suggest the function
as a measure of quality of alignment. In (8), R_{0} is an empirical parameter (chosen at 3 Å) that measures the relative significance of RMSD and . Computer experiments have shown that a square dependence on N_{align}/RMSD is as good as a cubic or linear one, and the second power was finally chosen only for technical convenience.
As seen from (8), Q reaches 1 only for identical structures (N_{align} = N_{1} = N_{2} and RMSD = 0), and decreases to zero with decreasing similarity (increasing RMSD or/and decreasing ). Therefore, the higher Q, the `better', in general, the alignment. Despite the fact that the Q score represents a very basic measure that does not take into account many factors related to the quality of alignment (the number of gaps and their size, sequence identity etc.), we found that maximization of the Q score produces good results.
In order to maximize the Q score of alignment, we first range all aligned pairs of C atoms by increasing interatomic distances: , . Unmapping of the most separated C pair decreases both the alignment length and RMSD. As may be found from the analysis of (8), such unmapping results in increasing Q at superlinear dependence R(i) and in decreasing Q at sublinear R(i). We therefore unmap C pairs one by one in order of decreasing interatomic distances until Q reaches a maximum (Q may change nonmonotonically). Owing to empirical considerations, we do not unmap inner atoms of mapped SSEs without first unmapping their outmost atoms, and, in matched SSEs, we never unmap n_{a} atom pairs with minimal separation (dark atoms in Fig. 3).
(v) Unmapping short fragments. Pairs of C atoms, which form short (1 or 2 pairs) closures between gaps, most often correspond to purely incidental intersections of protein chains. However, they may effectively lock the structures in a particular orientation and thus prevent further optimization. We therefore unmap such pairs even if doing so decreases Q.
The mapping obtained may be used for the calculation of best structure superposition by applying FOS to the pairs of mapped C atoms. Since a change in orientation may affect the mapping, the cycle mapping FOS is repeated until the Q score of alignment ceases to increase over a sufficiently large number of successive iterations (ten by our choice). The contact distance R_{c}, used for mapping atoms of nonmatched SSEs and in looking for contacts (cf. above) was found to be a very important parameter, which significantly affects the quality of results. In our implementation, R_{c} increases linearly from 3 to 5 Å during the first ten iterations.
The presented algorithm of C alignment converges to a local maximum of score function (8). Therefore, the results are highly dependent on the quality of the initial guess, which is provided by the identification of common subsets of SSE through the threedimensional graphmatching procedure. In the course of analysis of many individual matches, we have found that a larger common subgraph is not an absolute indication of a betterquality match. Therefore, for each pair of structures, SSM performs C alignment starting from all common subgraphs that are larger than N^{max}_{SSE}3, where N^{max}_{SSE} is the size of maximal common subgraph, and the alignment with the highest Q is accepted as a result. In our comparative study, presented below, we found that the overall procedure works very well if the structures show a reasonable degree of similarity. If structural similarity is very low (such that only one or two common SSEs may be identified), the procedure may result in a less accurate solution. In such cases, however, many imperfect alignments are usually possible, and choosing the best one is never selfevident.
4. Scoring the results
The score function Q [equation (8)] was found to be a good geometrical measure of structural similarity. As mentioned above, this function offers a compromise between contradicting requirements of achieving a lower r.m.s.d. and a higher number of aligned residues and, therefore, Q is expected to be a more objective indicator of quality of alignment than RMSD and alone.
However, higher structural similarity does not necessarily imply higher significance of alignment. For example, a helix may be perfectly aligned with most of the PDB entries, but the significance of such alignments is very low because they are likely to be obtained simply by chance, by choosing the structures randomly from the database.
Our estimation of statistical significance is based on the same ideas as those employed by VAST (Gibrat et al., 1996). The probability that matching two structures A and B is scored at value S or higher merely by chance may be estimated as the P value:
In this expression, P_{k}(S) is the probability of achieving the score S in the event when matching two structures, picked randomly from the database, yields a common containing k SSEs. M_{k} stands for the redundancy number, showing how many common substructures of size k may be formed from proteins A and B. We define score S as a sum of quality scores Q [cf. equation (8)] for the matched SSEs:
where index i numbers the matched SSE pairs, N_{A/B}^{(i)} is the number of residues in the ith matched SSE of protein A/B, N_{i} is the number of aligned residues in the ith SSE pair, and RMSD_{i} is the r.m.s.d. of the ith pair. Thus, for common substructures of size k, score S may vary from (poorest alignment) to k (ideal alignment). Definition (10) allows one to calculate P_{k}(S) as
under a reasonable assumption that scores Q_{i} do not correlate. In (11), is the density of the probability of finding a common containing k SSEs with score x by randomly choosing the structures from the database. The functions may be calculated for any k through their recurrent relation given by (11). This recurrence starts from the function , which is calculated empirically by running SSM on all pairs of nonredundant protein structures [we used SCOP folds as found in SCOP Version 1.61 (Murzin et al., 1995)].
The value of P_{k}(S), corresponding to the actual alignment, may also be of interest. SSM reports it as a Z score, defined by the following equation,
5. Implementation
The procedure described above for protein alignment in three dimensions has been implemented as a standalone application and as a web server, available for public use at https://www.ebi.ac.uk/msdsrv/ssm . The standalone application and the web server have identical functionality. The development is based on the new CCP4 Coordinate Library (Krissinel et al., 2004) and runs on all Unix platforms. SSM allows for a number of different tasks, including threedimensional alignment of protein pairs (uploaded or given as PDB/SCOP ID codes), alignment of a structure to all entries of the PDB/SCOP archives or any subset of SCOP, or alignment to an uploaded set of structures. For faster processing, SSM precompiles SSE graphs of all PDB and SCOP entries in fastaccess files, which are updated automatically on a weekly basis. Serial alignments (a structure to a set of structures) are automatically scheduled on a number of CPUs depending on the anticipated task complexity. Unlike many other protein comparison services, SSM does not keep a database of precalculated threedimensional alignments, because its performance was found to be sufficient for serving queries in real time.
A distinguishable feature of SSM is that its performance depends rather sharply on the minimal desired level of structural similarity set in advance. This feature follows from the properties of the original graphmatching algorithm that we employed (Krissinel & Henrick, 2004). The level of structural similarity is measured by the percentage of tobematched SSEs p_{SSE}. The higher p_{SSE}, the quicker is SSM. Typically, alignment of a fewhundredresidue protein to all PDB entries at p_{SSE} = 5070% takes much less than a minute.
6. Results and discussion
Fig. 6 shows a comparison of SSM with other publicly available web servers that deliver threedimensional alignment of protein structures, namely VAST (Gibrat et al., 1996), combinatorial extension (CE) (Shindyalov & Bourne, 1998) and DALI (Holm & Sander, 1993). The comparison is presented for the example of protein chain 1sar:A (ribonuclease SA; Sevcik et al., 1991). As seen from Fig. 6, all the servers reveal fairly distinctive subsets of highly similar structural neighbours (from the whole PDB) and structures with intermediate similarity. VAST also returns some dissimilar structures (as identified by the alignment length ). The number of returned hits differs from server to server, which probably reflects the different criteria used for the identification of insignificant matches. In SSM, we have chosen not to dispose of any hits found with similarity level higher than the requested p_{SSE}. Instead, SSM provides a facility to sort the results by a variety of scores [Q score (default), P value, Z score, RMSD, , number of matched SSEs, number of gaps etc.] and tools for navigation through the matches. This approach was motivated by the consideration that none of the scores provides an absolutely reliable measure of structural similarity or statistical significance, and therefore the final decision of accepting a match should be reserved for the user.
For the calculations presented in Fig. 6, we used p_{SSE} = 0; therefore, SSM produced tens of thousands of matches, from which we present only those also returned by VAST, CE and DALI in Fig. 6. It appeared that SSM has found all the structural neighbours found by the other servers, with the exception of a few structures represented by C atoms only. Such structures do not allow for the determination of hydrogenbond patterns and, consequently, for a reliable calculation of secondary structure. Many (from tens to hundreds) hits in the similarity range of Fig. 6, returned by SSM, are not found in other servers' outputs. Although it is difficult to explain this result without knowing all details of the algorithms and their implementations, a higher fraction of newer structures in SSM results suggests that the difference is partially due to outdated databases. Other reasons may include narrowing of the search by different similarity criteria and use of representative structures instead of actual screening of the whole PDB.
Fig. 6 suggests that SSM fully agrees with the other servers in the identification of highly similar, less similar and dissimilar structures. The alignment length shows clear stepwise `transitions' between the subsets of structures with different structural similarity to 1sar:A . In this particular comparison, SSM alignments are longer than those given by VAST, somewhat shorter than those from CE and of approximately the same length as those of DALI. A thorough examination of plots for RMSD and reveals that longer alignments always come at the expense of higher r.m.s. deviations, and therefore the observed differences between the servers should be mostly due to the different criteria employed to balance these characteristics. This conclusion is corroborated by the observation that all servers agree on the r.m.s.d. for highly similar structures, when all residues of 1sar:A are aligned to targets.
The balance of RMSD and is indicated by the Q score [equation (8)]. As seen from the Q score plots in Fig. 6, all servers deliver threedimensional alignments with very close values of Q. Although the Q scores differ in value (with the largest difference found between SSM and VAST, while Q scores from SSM and DALI nearly coincide), they show a very good correlation, which is seen in that the Q curves are much smoother than the corresponding curves of RMSD and . Fig. 6 also suggests that the Q score allows for a clearer identification of highly similar and less similar structural neighbours than do RMSD and alone. A particular example can be found in the SSM–CE comparison (column b in Fig. 6). Judging by the alignment lengths obtained from CE, PDB entries numbered 120–220 may be qualified as closer structural neighbours to 1sar:A than entries numbered 30–119, which contradicts the SSM findings. Analysis of the corresponding RMSD plot suggests that the above conclusions may be incorrect. If, however, the Q score is taken as a measure of structural similarity, SSM and CE agree on rating the entries numbered 120–220 below those numbered 30–119. Since the Q score takes both RMSD and into account, the last conclusion seems to be more justified. Another example of this kind may be found in the VAST results for structural neighbours numbered 220–265, showing a decrease in both and RMSD, which therefore may be interpreted as either a decrease or an increase in structural similarity. At the same time, and RMSD obtained from SSM unambiguously suggest decreasing similarity for these structures. A clear fall of Q scores obtained from both servers implies that the structures are, indeed, ordered by the decrease of their similarity to 1sar:A .
We performed a comparative study, similar to that described above, for a number of structures belonging to different protein folds. The results shown in Fig. 6 were found to be of a common nature. More results are published on the SSM web site.
The results obtained suggest that different similarity scores perform equally well if structure similarity is high. The situation, however, changes drastically as the similarity decreases. For example, PDB entry 1kn0:A (human RAD52 protein; Kagawa et al., 2002) does not have an exact match in SCOP Version 1.61. All potential matches to 1kn0:A from SCOP 1.61 domains represent relatively remote structural neighbours, and therefore SSM should be run with a low similarity threshold. Choosing p_{SSE} = 15% results in a total of 33 588 hits returned, none of which represents a perfect match. Fig. 7 shows superpositions of 1kn0:A with bestmatching SCOP domains, as suggested by Q score, RMSD, and Z score. The achieved scores are presented in Table 2.

As seen from Fig. 7, the highest Q score indicates a match (Fig. 7a; d1di2a_; doublestranded RNA binding protein A; Ryter & Schultz, 1998) that is (geometrically) best according to common intuition. Although the overlap is not perfect, the common substructures are compact and form most of the target structure. The matches with the lowest r.m.s.d. (Fig. 7b) and highest Z score (Fig. 7d) represent alignments that are too short to be rated high. The match with the maximal number of aligned residues (Fig. 7c) shows a poor superposition of common substructures with high r.m.s.d.; the alignment is fragmented and the overall overlap seems to be incidental.
The results show that using an appropriate score is crucial for the similarity search. An idea of what it would take to find d1di2a_ as the best match to 1kn0:A without using the Q score may be obtained from Table 3. The table lists the ten best matches, all of a comparable quality, rated by different scores. As may be seen from Table 3, d1di2a_ is 1575th by RMSD, 3079th by Z score, 818th by P value and 872nd by alignment length (since is an integer number, the last figure is subject to the sorting procedure). Thus, d1di2a_ does not appear on top of result lists sorted by any of the traditionally used similarity scores, and it would take many hours, if not days, to find this match manually from the results.

It is commonly assumed that protein chains with similar sequences tend to fold into similar threedimensional structures. This assumption is often used for narrowing the similarity search or for the selection of representative structures. Although using the assumption makes the search faster, a known side effect is that the results may be biased toward sequence similarity. Because our alignment procedure is completely indifferent to chain composition, we used SSM for studying the relationship between sequence and structure similarity. Fig. 8 shows correlations between sequence identity (SI), Q score, RMSD and the normalized alignment length N_{m}:
The sequence identity is defined as a fraction of identical residues in the total number of (structurally) aligned residues:
The score correlations are represented by contour maps of the reduced density of the probability, , of obtaining threedimensional alignment with particular values of scores x and y:
where probability density is calculated in the course of alltoall alignment of all chains found in the PDB.
As seen from Figs. 8(a)–8(c), 100% sequence identity does not necessarily mean a perfect threedimensional alignment in terms of either Q score, RMSD or alignment length. Values of at SI = 1 (Fig. 8b) indicate pairs of chains with sequenceidentical common subchains. Despite the absolute sequence identity, these chains show structure differences with an r.m.s.d. of up to 1 Å (cf. Fig. 8c). Most of these differences are caused by the interaction between residues of matched and unmatched parts of the chains, and therefore 1 Å of deviation per of difference in chain length may be considered as a measure of that interaction or as an effect of chain length. In order to estimate the effect of chain composition on its threedimensional structure, consider matches with N_{m} = 1. The value of N_{m} = 1 corresponds to fullchain alignment and therefore indicates highly similar threedimensional structures. As seen from Fig. 8(b), having as few as 20% of identical residues is already enough for chains to fold into highly similar structures. This conclusion generally agrees with previous findings (Chotia, 1992; Chotia & Lesk, 1986; Hubbard & Blundell, 1987). Comparison with Fig. 8(c) suggests that the difference in structure increases quite regularly with decrease in sequence identity, reaching 1–2.5 Å at SI ≃ 20%. The decrease in structure similarity is seen as an exponentiallike increase in RMSD, which has also been found in other studies (Chotia & Lesk, 1986; Hubbard & Blundell, 1987; Flores et al., 1993; Russell & Barton, 1994; Russell et al., 1997). Thus, the well defined ridge of the RMSD plot at in Fig. 8(c) represents the effect of chain composition on the threedimensional structure of similar chains.
Structures with less than 20% sequence identity show a wide range of RMSDs and alignment lengths, while Q does not reflect this effect (with the exception of a few `islands' at intermediate Q and SI). Fig. 8(d) demonstrates a clear reduction of the correlation between RMSD and N_{m} at Å and , which region, as may be derived from comparison with Figs. 8(b) and 8(c), corresponds to %. These results lead to the conclusion that % is a solid indication of low structural similarity, when reliable detection of common submotifs is not feasible. Usually, more than one common with very close values of Q may be identified between remote structural neighbours. Then, alignment of structure A to its remote neighbours B and C is likely to lead to the result that the best common for A and B is not the same as that for A and C, even if B and C are highly similar (but not identical). This uncertainty in the detection of common substructures arises due to small variations of Q at small variations of SI, and therefore the correlation between Q and SI should not be affected. However, close values of Q for different common substructures do not imply closeness of the corresponding values of RMSD and . Simple considerations show that at lower structural similarity, the RMSD and values of common substructures with close values of Q (and, consequently, SI), may show a wider range of variations. Therefore, with decreasing structural similarity, the correlation between RMSD, and SI should vanish. This is exactly the picture seen in Fig. 8 at %.
As shown by the obtained results, RMSD is a good score if the structure similarity is sufficiently high that more than 80–90% of residues are aligned. This situation corresponds to structures with obvious similarity, for which RMSD gives merely a measure of distortion. The alignment length does not perform well at any degree of similarity, and allows only for a rough indication that 80–90% of aligned residues correspond to highly similar structural neighbours. The Q score performs more or less uniformly in the whole similarity range, except for a few islands aside of the main ridge in Fig. 8(a). It is therefore expected that the Q score should be particularly useful if structural similarity is not obvious. This assumption is fully confirmed by the above example of 1kn0:A , which falls into the `nonobvious' category, judging by the values of SI shown in Table 2. We have performed a series of experiments on the comparison of remote structural neighbours, which have convinced us of the above conclusion.
Consider now the relationship between the structure/sequence similarity and the statistical significance of the matches (Fig. 9). Since statistical significance depends on both the similarity of matched structures and the composition of the database, a perfect match does not necessarily correspond to the lowest P_{v} and highest Z. As may be seen from the figures, this is, indeed, the case, and at , , a wide range of P_{v} and Z values are attained. Although, on average, statistical significance increases with increasing structure/sequence similarity, the correlation decreases significantly at higher Q and SI [note that the effect of Z should be estimated through integral (12), and the significance of a hit changes in inverse proportion to P_{v}]. Therefore, statistical significance scores are very sensitive to small structural variations between close structural neighbours, being nearly indiscriminative if structural similarity is low. These findings agree with intuition. Indeed, one expects to find no more than one structure, identical to the query (Q = 1), in the whole PDB, which finding is then a highly significant event. However, that structure's fold or family will normally have a considerable number of highly similar structural neighbours, even with Q just slightly lower than 1. These matches will not be very surprising in statistical terms. Hence the difference in statistical significance of hits to similar structures with should be high. Conversely, detection of low similarity is statistically insignificant, no matter how exactly dissimilar, in one of many million ways, the structures are. Therefore, small differences in correspond to relatively small differences in log(P_{v}) and Z.
Values of and indicate hits that are completely expectable, for example, finding a structure containing a helix or a strand. The Q score of such hits does not exceed 0.3 at , which corresponds to low structural similarity. As seen from Fig. 9, the region of low similarity is bounded by . This fact has a simple explanation as the nonredundant database, which we used for the calibration of P values [that is, the calculation of , cf. equation (11)], was composed of folds of SCOP 1.61. Therefore, nontrivial matches are expected to emerge with probability lower than 10^{2.8}.
Comparison of Figs. 9(a) and 9(b) with Figs. 9(c) and 9(d) shows that the Q score correlates with statistical significance better than with sequence identity. The overal difference in the landscapes is explained by the relationship between Q and SI in Fig. 8(a), which shows that Q is not sensitive to SI at . At the same time, it is curious enough to see that, with the exception of a few islands in Fig. 9(c), the P value does not show any evident dependence on chain composition at .
7. Conclusion
More than two years of working with SSM and studying the feedback from its users worldwide has convinced us that SSM represents a powerful, flexible and accurate tool for protein structure comparison in three dimensions. It is particularly efficient, as compared with other similar resources available, when applied to large protein structures (more than a few hundred aminoacid residues) and for matching a structure to a precompiled database of structures (PDB, SCOP or userdefined).
The competitive performance of SSM is mostly a result of the original graphmatching algorithm employed (Krissinel & Henrick, 2004). In the present study, we did not compare the efficiency of SSM with that of similar algorithms, although, in our experience, SSM is at least an order of magnitude faster. However, a direct and objective comparison is hardly obtainable. Many other services are not interactive, which prevents direct time measurements. Most of the existing services maintain a database of precalculated alignments or use sets of representative structures, so that the number of actual alignments is never the same. Finally, SSM runs on a CPU cluster, employing different numbers of CPUs depending of the task complexity, while little is known about the implementation and hardware basis of other developments.
The iterative procedure of C alignment as described in this paper includes a number of empirical elements and parameters. These elements were introduced and the corresponding parameters tuned in the course of analysing of thousands of alignments. As a result, comparison of SSM with other similar servers shows a good overall agreement, to the degree of difference between all of them.
Because of the evergrowing number of solved protein structures, automatic recognition of their structural motifs becomes an increasingly important task. The very definition of structural similarity remains, however, a vague issue in general. Unless the similarity is selfevident, there is no perfect quantitative measure for drawing a line between similar and dissimilar structures, and even for ranging structure pairs in order of their similarity. Because of this circumstance, any test on true/false positives/negatives is never fully convincing, and therefore such a test was omitted in the present study. In the numerical study presented in this paper, we considered a few scores applicable to measuring the structural similarity. As shown, the most obvious scores of RMSD and alignment length do not provide a sufficient level of confidence in structure recognition. The best quality of structure recognition is achieved by using the introduced Q score [equation (8)], which combines both RMSD and the alignment length. The Q score represents a measure of quality of threedimensional alignment and is maximized by the SSM's C alignment algorithm. Although the Q score should be viewed only as a model simplification of an intuitive understanding of the alignment quality, we found that in practice it works very well. It should be noted that there are other scores combining the alignment length and relative remoteness of aligned residues (see e.g. Russell & Barton, 1992; Kleywegt & Jones, 1994), which we did not investigate in this study.
APPENDIX A
Fast optimal superposition in three dimensions
A number of methods have been reported for the calculation of the rotation matrix , which optimally superposes two sets of points in threedimensional space, and , , such that (both sets are brought into their centres of mass)
(w_{i} are weights) is minimal (see e.g. McLachlan, 1972; Kabsch, 1976, 1978; Lesk, 1986). The methods involve converging iterations, diagonalization or orthogonal decomposition of the correlation matrix (Lesk, 1986),
We found that the best results are obtained using singular value decomposition (SVD), which is a very stable numerical procedure applicable even to singular correlation matrices. According to Lesk (1986), , where is a (unique) Hermitian positive definite matrix. Applying SVD to matrix , we obtain
where and are orthonormal matrices and is a diagonal matrix of (always nonnegative) singular values. Considering that represents a Hermitian positive definite matrix, we obtain
This procedure, however, does not guarantee that will represent a proper rotation. If then the superposed set is inverted (rotoinversion) (Kabsch, 1978). There is no way out of this problem other than to make an appropriate correction to the correlation matrix . As follows from equation (19), changing the sign of any of the vectors or will change the sign of and thus make the matrix of proper rotation. Such a change of sign is equivalent to a distortion of . Since (Lesk, 1986)
such a distortion may result in increasing D. As may be derived from equations (18) and (20), this increase is least (and therefore the resulting proper rotation is the best possible one) if changing the sign is applied to the vector or that corresponds to the minimal singular value .
It is important to note that the calculation of the rotation matrix using SVD gives a meaningful result even if the correlation matrix is degenerate, which fact was taken into account in our choice of method. The optimal superposition is achieved by applying the rotation matrix to structures {x_{i}}, {y_{i}} brought into their centres of mass.
Acknowledgements
The authors are thankful to Dr Stephen H. Bryant for a detailed explanation of the P value calculations in VAST (Gibrat et al., 1996). EK is grateful for support from the BBSRC Collaborative Computational Project No. 4 in Protein Crystallography (Collaborative Computational Project, Number 4, 1994).
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