GroES-ATP7-(single-ring)GroEL [29] to GroES-ATP7-GroEL [28]. These complexes were determined at different resolutions (see Table 2) and have different compositions; the first object in each pair is a substructure of the second. In both tests correct matches were ranked at the top despite the different assembly sizes and differences in conformation. The DifferenceGrid results for the GroEL-ATP7 and GroES-ADP7-GroEL-ATP7 pair highlight the conformation differences upon ATP binding to the GroEL cis ring (see Figure 2B) whereas the results for the GroES-ATP7- GroEL and GroES-ATP7-(single-ring)GroEL pair suggest that these two complexes are similar. The matching of the different conformers of TClpB provided another test for the influence of conformation changes on the success rate of our procedure. TClpB is an ATP-dependant molecular chaperone which disaggregates stress-damaged pro- teins. Its structure consists of two hexameric rings (AAA-1 and AAA-2) rotated by 23u about the co-axial 6-fold rotation axis. It was suggested that the exposed face of the AAA-1 ring binds the substrate which is then threaded through the central pore [10]. The rings are of similar size in the ATP bound TClpB mutant (E271A, E668A) but not in apo TClpB. We note that in the self- matching of the ATP bound complex our algorithm identified a pseudo-symmetric match in which the rings interchange (rotation of 180u about an in-plane axis plus 24u about the 6-fold axis). The score of this (false) match is however only 22% of the score of the correct match. The self-docking of apo TClpB did not produce such a pseudo-symmetrical model. We matched the ATP bound TClpB mutant and the AMPPNP and ADP bound hexamers to each other. In all three cases correct matches, similar to the manual fittings [10], were ranked at the top. The ATP- and ADP-bound TClpB matching was executed employing rotational intervals of 8, 12 or 16u, in order to test the sensitivity of the results to this parameter. A correct match was ranked at the top in every case; the scores of the top ranking models obtained in the 8u and 16u searches were slightly higher than the score obtained in the 12u search, indicating that a relative rotation of 4u marginally improves the match. This result indicates that the 12u rotation interval is adequate for identifying the correct match; yet, top ranking matches should perhaps be refined by testing small local rotations from the predicted position. The difference between the grid representations of AMPPNP- TclpB and the top ranking matched ATP-TClpB, shown in Figure 2C, is in line with the observations of Lee et al. [10]. It highlights the extended spokes in the TClpB-AMPPNP complex compared to the shorter and thicker spokes in ATP-TclpB. There is additional density on top of the upper ring in the ATP-TClpB mutant complex and in the central pore region, making the pore narrower. The differences in the shapes of the lower ring of the two objects are minor and may be related to errors in the EM maps and in the matching. The radial spokes are even shorter in the ADP-TclpB complex and the central pore of the upper ring is narrower, in particular in the lower half. We note additional structural differences at the interface between the rings. Two additional examples for matching of different conformers of the same molecule are the closed and open structures of the human transcription factor TFIID [30] and the apo and ATP bound Drosopila origin recognition complex (ORC) [31]. In these cases we also tested the effect of small changes in the density cutoff of the EM map on the matching results. We therefore gradually increased the volume of object A (by slightly decreasing the density cutoff value) and decreased the volume of object B (by increasing the density cutoff value). A correct match was ranked at the top in all the scans however the complementarity appeared to be better (higher scores) as the density cutoff of object A was reduced and Matching & Docking EM Maps that of object B was increased. Notably, in both examples the EM map grid intervals and consequently the grid interval used in the FitEM2EMIN matching, are particularly large (.5 A˚) but the identification of correct matches was not affected. Finally we matched the 40S subunit of the human ribosome [32] to the whole ribosome (80S) assembly formed by the 40S and 60S subunits. The effect of the density cutoff change in this case was the same as described above. Thus the position of the 40S subunit within the 80S structure in the top ranked match did not change. The complementarity score however gradually increased as the density cutoff value for the 80S structure was decreased and that of the 40S structure was slightly increased, indicating that the shape and size of the 40S subunit match better within the 80S ribosome. The DifferenceGrid results (see Figure 2D) for the top ranking match can be used to approximate the shape and size of the ribosomal 60S subunit, producing a similar result to the subunits separation presented by Sphan et al. [32]. Docking EM maps Docking of low resolution objects is important in assembly modeling and the program FitEM2EMOUT was designed to perform such tasks. We could not find EM maps of substructures whose overall structure is known in order to calibrate the parameters of FitEM2EMOUT and test the program. Therefore, the same parameter values and grid representations as described for FitEM2EMIN were employed and simulated electron density maps were docked to EM maps. The simulated maps were calculated using the pdb2vol and map2map modules of Situs [19]. The electron density cutoff for the simulated map was chosen to encompass all the atoms in the corresponding PDB structure. In the first test, the coordinates of the trans ring of GroEL [33] from PDB [34] entry 1AON were used to calculate an electron density map at 10 A˚ resolution (the resolution of the docking partner). This map was docked to the EM map of GroES-ATP7- (single-ring)GroEL complex [29]. Several symmetry related correct matches were ranked at the top followed by a false match (ranked 5) in which the tip of GroES makes contact with the central void of the GroEL trans ring. Fitting of an extreme value distribution function to the distribution of scores [35] provided estimated E values of 5.661024 and 2.161023, respectively, for the highest ranking correct and false models. The second test involved docking of a simulated map of the tetramer of the oxidoreductase b2 subunit [36] (PDB entry 1EXB) to the EM map of the 4:4 complex between the pore-forming Kv4.2 subunits and the regulatory KChlP2 subunits [37]. The resolutions of both the EM map and the simulated electron density map were 21 A˚. The b2 surface that faces the T1 domain of the pore forming subunit in the T14-b24 complex [36] is expected to make contact with Kv4.2*-KChlP2 [37]; indeed this general position of the simulated map of b24 is observed in many high ranking docking models. These models differ in the rotation angle of the b2 tetramer about the 4-fold symmetry axis and the docking scores change periodically as function of this angle (see Figure 2E). The score difference between the best and worst positions is within 3s. The highest scoring false model, in which the symmetry axis of b24 is not aligned with that of Kv4.2*-KChlP2, is ranked 35. The E values for the highest ranking correct and false models are 5.1261026 and 1.3961023, respectively. Discussion The recent upsurge of low resolution structures obtained by different experimental techniques and the capability to study global structural changes by cryo electron microscopy, introduced PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3594