ARP/wARP and molecular replacement: the next generation
Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information.
With the advent of structural genomics initiatives (Stevens et al., 2001) and more focused high-throughput structure-determination projects (Banci et al., 2006), the need for advanced methods for structure determination has been emphasized (Lamzin & Perrakis, 2000). A specific research investment in automatic model building has led to significant developments; the advances of software such as RESOLVE (Terwilliger, 2004), TEXTAL (Ioerger & Sacchettini, 2003) and Buccaneer (Cowtan, 2006) are some prime representatives of this effort. These programs are typically based on interpreting electron density directly in terms of small secondary-structure elements and extending the model from such seeds. Typically, the input is a set of structure-factor amplitudes and some phase estimates or phase probability distributions.
Conversely, the ARP/wARP software suite (Morris et al., 2004) provides a `pipeline' which unifies model building with model refinement. The electron-density map is parameterized with a set of representative `free' atoms. After initial model building, once a fraction of these atoms acquires a chemical identity the `hybrid' model (consisting of free atoms and atoms with known stereochemistry) is iteratively refined and edited, taking advantage of the improved electron-density maps produced by refinement (Perrakis et al., 1999). A main advantage of the iterative editing performed by ARP/wARP is the ability to recycle newly derived chemical information into a better estimation of the electron density. A direct effect of this approach is that models from molecular replacement are directly incorporated into the ARP/wARP `pipeline'. As ARP/wARP is centred on the use of an atomic `hybrid' model, handling of models derived from molecular replacement is particularly well suited to the ARP/wARP formalisms.
As the number of structures deposited in the PDB increases, molecular replacement is more and more likely to be the method of choice for structure solution. Many efforts are currently under way to enable molecular replacement to work with search models that are either partial (especially in the context of macromolecular complexes) or have low identity to the structure in question.
The molecular-replacement method for crystal structure determination has developed significantly in the past two decades. AMoRe (Navaza, 2001) led the way to automation and addressed the need to perform all the core tasks (rotation function, translation function and rigid-body refinement) within calls to tightly unified program modules. As experience accumulated, both AMoRe and other software increased in sophistication and required fewer decisions by the end user. For example, MOLREP (Vagin & Teplyakov, 2000) included a packing-function term in its search target and an automatic search for multiple copies of the same protein in the asymmetric unit. More recently, more accurate search-target functions have been developed; the program Phaser (McCoy et al., 2007) was recently developed to implement a molecular-replacement search using an approximation to the crystallographic maximum-likelihood target function; furthermore, Phaser automated complex search strategies. Meanwhile, more effort has been made in the automation and integration of the preparation of search models. For example, CHAINSHAW (N. D. Stein, work to be published) uses an alignment between the sequence of the protein in the crystal and in the search model to edit the latter (pruning long side chains and deleting unconserved residues). In a similar spirit, the web application elNémo (Suhre & Sanejouand, 2004) implements normal-mode analysis to sample possible alternate conformations of the search model before starting the molecular-replacement search. More recently, there has been an effort to further integrate existing software: for example, MrBUMP (Keegan & Winn, 2008) and BALBES (Long et al., 2008) integrate in different ways the identification and editing of search models with the actual molecular replacement and assessment of the quality of results.
After describing the ARP/wARP workflow in the context of a molecular-replacement solution, we present two specific case studies. These are used to build an understanding of how the model evolves during the ARP/wARP iterative process, as implemented in the flex-wARP control system. The core of the article deals with the systematic study of 129 cases of automatic molecular-replacement solutions for which a corresponding reference structure is available: after evaluating the relative success rate of all three flex-wARP standard protocols, we use the gathered statistics to build an empirical estimator that predicts the quality of the outcome of flex-wARP (how good the final map and how complete the final models produced by flex-wARP are likely to be) based on the a priori known experimental data and the quality of the molecular-replacement solution.
The ARP/wARP general workflow has been described in Perrakis et al. (1999) and specific issues relating to molecular replacement have been presented in Perrakis et al. (2001). Here, we briefly discuss the general principles and our renewed experience of molecular-replacement protocols, highlighting features specific to the new control system flex-wARP.
ARP/wARP typically starts by building a model made of `free' atoms in order to represent the electron density. All further steps involve the interpretation of the coordinates of the atoms present in the current model and the current electron-density map. The steps taken by ARP/wARP are as follows.
In the following examples and discussion we use a new control system for the ARP/wARP process, flex-wARP (previously also introduced as pyWARP). Flex-wARP parses the output of the process modules in order to decide at run time what is the action to be taken next: the outcome of every decision is which actions have to be taken and which decision these actions lead to when completed. This concept, described briefly in Cohen et al. (2004), can be represented as an oriented graph, in which arrows represent actions and nodes stand for decisions (Fig. 1). As described in the following sections, this graph can be entered at different decisions/nodes when the structure is solved using molecular replacement. A fundamental difference from the `classic' ARP/wARP scheme is that the number of refinement steps (internally in REFMAC but also between autobuilding cycles) as well as the total number of cycles are not pre-decided but are dynamically defined according to model evolution. In addition, when the model is complete enough all free atoms are deleted and only waters are added until convergence. Moreover, a novel loop-building algorithm (Joosten et al., submitted) joins main-chain fragments that are separated in sequence by 14 or fewer residues.
Molecular replacement positions the search model in the correct place in the asymmetric unit. The model is then directly subjected to iterative model refinement, update and rebuilding. The main advantage of this procedure is that it keeps all chemical information contained in the search model (in terms of stereochemical restraints) and provides more information to the initial refinement stage before the first automated model building takes place.
In most cases, it is beneficial to use the restraints provided by the molecular-replacement search model for the first refinement steps. However, if most of the restraints are genuinely invalid (e.g. when a very poor starting model is used) it may be beneficial to remove the chemical identity of atoms by simply keeping the atomic coordinates but turning all atom types to free atoms, `DUM', effectively eliminating all stereochemical information. This free-atom model is refined and updated without restraints until the first automated model-building step that will assign new chemical identity.
The positioned search model can also only be used to compute an initial electron-density map, which is then interpreted in terms of a free-atom model (as for any other experimental map). This model is improved by a few cycles iterating refinement and model update. A first model is then built and the process iterates all three actions. It is clear that this procedure is not using any of the chemical information that is contained in the molecular-replacement model; hence, it will rapidly remove most of the bias introduced by the molecular-replacement model.
In the next part we will use two specific examples to illustrate how ARP/wARP iteratively edits and optimizes the model output from molecular replacement, leading in these cases to highly complete and accurate structures. We will then report the medium-scale systematic study that was required to draw statistically sound conclusions about the usefulness of the application of ARP/wARP procedures to molecular-replacement solutions.
The first example illustrates how ARP/wARP can complete a partial molecular-replacement model. It concerns the solution of the structure of a noncovalent complex between Ubc9 (an E2 ubiquitin ligase) and SUMO (a ubiquitin-like regulator of Ubc9 in the context of this complex; Knipscheer et al., 2007). A data set at 1.8 Å was used for this study. Molecular replacement was performed using only Ubc9 as the search model, representing only two-thirds of the ordered asymmetric unit content. The model was directly input to flex-wARP using the default protocol; that is, keeping both atom coordinates and chemical information during the first update cycles (as described in §2.2). The evolution of the quality and completeness of the automatically built model is summarized in the top two panels of Fig. 2.
Within 40 steps of refinement and model update, the number of atoms reaches the expected number for the expected asymmetric unit content. This in turn enables the model-building algorithms to build both Ubc9 (which was already provided by the molecular-replacement solution) and SUMO. After step 70, when the flex-wARP decision system considers the model to be of good quality, final model clean-up and re-arrangement takes place: the number of atoms decreases sharply (since all free atoms are removed) and over the next ten steps water molecules are added to model the ordered part of the solvent. At the same time the loop-building algorithm joins a few chains and builds extra residues with their corresponding side chains.
We also tested the two other protocols: starting from the coordinates only or the electron-density map (as described in §§2.3 and 2.4, respectively; middle and bottom panels of Fig. 2). Both tests led to fairly complete models of quality similar to that built using the standard protocol. However, this required 110 and 73 steps, respectively, compared with 81 steps for the default protocol. The faster convergence of the protocol in which all expected free atoms are placed in electron density first is expected since the molecular-replacement solution was a partial model. Finally, since in these two cases restraints take a few cycles to accumulate through model building, the initial steps of refinement show strong overfitting (maintaining up to 22% and 19% difference between Rwork and Rfree, respectively), whereas the largest difference between Rwork and Rfree, when starting from the search model and maintaining stereochemistry, is 10% (with a mean difference of 8%).
The second example shows how ARP/wARP can help to recover a very poor molecular-replacement solution. It is based on the high-resolution structure determination of the SMR domain of the MutS2 protein from Helicobacter pylori (Lebbink, Radicella & Sixma, work to be published). Diffraction data were collected from a crystal diffracting to a resolution of 1.0 Å and containing two copies of the protein in the asymmetric unit. The structure was solved by molecular replacement using an NMR model with 19% sequence identity as search model. Despite the high resolution and a fairly clear molecular-replacement solution provided by Phaser, the positioned model did not refine and the resulting electron density was hard to interpret (as reflected by a map correlation of only 32% with the reference model).
The evolution of the iteratively rebuilt model using the standard protocol (as described in §2.2) is shown in the top panels of Fig. 3. As the model is edited and completed (many atoms were missing after the editing of the search model), both Rwork and Rfree rapidly decrease. This corresponds to a sharp increase of the map correlation to the reference. After step 25, only minor editing takes place and the final model (with 160 out of 168 residues) is obtained at step 60.
We again tested the other two protocols we describe on this data set. Starting from coordinates alone (removing the restraints, as described in §2.3; middle panels in Fig. 3) works well but takes more time (70 steps instead of 60), which is somewhat surprising; restraints should not matter much when 1.0 Å data are available. Also surprisingly, starting from the map generated by the molecular replacement (protocol described in §2.4; bottom panels in Fig. 3) proved to require a very large number of steps to be able to produce useful results: the first side chains are built at step 297 and flex-wARP considers the model to be complete only after step 391. This could be explained by the poor quality of the initial map resulting in wrong positioning of the seeding atoms; somehow the atomic information of the search model contributes very significantly despite the very high resolution of the data. Because ARP/wARP peptide recognition is optimized for a resolution in the range of 1.6–2.5 Å, we also applied the protocol described in §2.4 after cutting the data to a resolution of 1.4 and 1.6 Å. This did not lead to any improvement in convergence speed or model quality.
The previous two examples were used to show how the model evolves during automatic iterative model rebuilding, especially in the context of the new ARP/wARP control system flex-wARP. To reach statistically sound conclusions concerning the benefits of using each of the three proposed protocols (§2) requires a large number of test cases. Here, we present the results of a medium-scale systematic study based on 129 deposited structures.
A large number of statistical indicators are available to evaluate the quality of the produced model when the final result is not known: Rwork, the figure of merit, the likelihood gradient (during refinement), the number of built atoms etc. Since all these indicators are strongly correlated to each other, we chose to show only the fraction of built residues (the number of residues in the final model divided by the expected number of residues given the sequence information and the number of copies in the asymmetric unit) and the fraction of docked residues (the number of residues which were assigned to the sequence and for which the side was built divided by the expected number of residues).
Additional to the above indicators, when a reference model for each data set is available for test reasons, additional metrics can be employed correlating the result of the automated process to the reference set. From these indicators we chose to show the correlation between the reference electron-density map and that obtained from the final model.
For the sake of clarity, for graphical representation we pool the data sets according to the `initial R factor' and to the `resolution' of the diffraction data, instead of crudely plotting results for each of the 129 data sets. The `initial R factor' is the Rwork produced by the positioned search model prior to any positional/ADP refinement.
Many other indicators are available and it is also valid to group the data sets in different ways for presentation; the primary data for all tests are available as a set of tables in ASCII text files from https://xtal.nki.nl/~serge/BALBES-1 for the benefit of the curious reader.
The default protocol (§2.2; Fig. 4) shows that when the initial Rwork is better than 30% automatic model building is likely to produce useful results. Conversely, a molecular-replacement solution producing an Rwork of between 30 and 40% is almost equally likely to be rescued by the default flex-wARP procedure or fail to produce results of any use; however, there is a tendency to improve the map quality (as shown by the values of map correlation) but produce fairly incomplete models. When success is assessed as a function of resolution, the fundamental tendencies of ARP/wARP show up: when data better than 2.0 Å are available, ARP/wARP fails only occasionally (presumably when the starting model is really very bad). Between 2.0 and 2.5 Å models are in general less complete and more cases tend not to work, but in general the runs are successful. With data weaker than 2.5 Å there are occasional successes that produce models close to 80% completeness, while below 3.0 Å we did not observe a single successful case. These observations are well correlated with the general ARP/wARP success rates, but also show that ARP/wARP can often produce good model-building results even from data that do not extend beyond 2.5 Å.
The two alternative protocols (§§2.3 and 2.4; Fig. 5) usually produce poorer results than the default. Nevertheless, there are a few exceptions where these alternate possibilities were more successful. Starting from atomic coordinates (§2.3) occasionally works better, but there is no clear tendency. However, it is notable that one case that shows 40% more docked residues built than the default protocol is at rather low resolution (2.5 Å) and has a relatively high starting Rwork (35%); we performed a detailed visual inspection of the initial (molecular-replacement) model and the reference model but were unable to derive a straightforward explanation for the behaviour of this particular data set. The third protocol (§2.4) does not show any advantage over the other two in general. However, it yields significantly better results when the starting model is bad (as indicated by Rwork) and the resolution is poorer than 1.5 Å; the difference can be as much as 40% more residues, with 20% more residues being quite often the case. These results could be further enhanced if advantage of NCS averaging or density modification was taken before such runs, but unfortunately the benefits of such a `pre-treatment' of the electron density before starting the model rebuilding could not be systematically tested here.
Having a reference structure, an objective assessment of the quality of the model built by flex-wARP can be derived. However, in normal day-to-day use the program is obviously run without knowing the result and, having the benefit of hindsight, knowing the reference structure. In other words, assessing the quality of the electron-density map produced by flex-wARP is not as trivial as computing a correlation with the 'final' reference map. It would be useful to know whether it is possible to use parameters available immediately after successful molecular-replacement solution to predict the quality of the map that flex-wARP will produce and ultimately the success of the ARP/wARP procedure. Using the relatively large number of test cases presented in this study, we tried to build an estimator of the produced map quality; the final map quality correlates well with the percentage of automatically built residues.
Of the large number of parameters which are available after the molecular-replacement solution, most were found to be of little predictive value or were redundant with the final parameters we chose: the initial R factor (RMOLREP), the high-resolution limit of the data (Resolhigh) and the solvent content (SC). We developed a good-quality estimator using these parameters and an intercept (constant term) with the following formula,
The quality of the estimator is shown in Fig. 6, which shows a scatter plot of the estimated value at the start of the run against the final `true' value computed against the correct reference. The proposed formula is clearly a crude estimator of the final results of flex-wARP, but nonetheless it can prove useful to quickly estimate the quality of an ARP/wARP run starting from a molecular-replacement solution. Upon detailed observation of the current mathematical model one sees that its main defect is the inability to express saturation; the target value (map correlation to the reference model) is bounded and the bounds are reached even for non-extreme cases. Hence, the response function is likely to be better modelled by some sort of sigmoid function instead of a linear one; unfortunately, this type of model requires more training data than are currently available.
The data used in this study were collected to a resolution of 1.80 Å. The structure was solved by molecular replacement using the program Phaser; the search model consisted of the structure of Ubc9 alone (PDB code 2grn , 158 residues), which corresponded to only two-thirds of the expected ordered content of the asymmetric unit. Hence, one-third of the structure, the SUMO protein consisting of 79 residues, was missing from the model and still had to be built. The model resulting from molecular replacement produced an Rwork of 46% and was directly input to flex-wARP. The models obtained after each side-chain-building step were compared with the reference structure in order to be able to assess the evolution of the map and model quality.
As a reference structure we used a structure of the same complex solved by molecular replacement and refined against 1.4 Å data in a slightly different crystal form (PDB code 1u9a ). The difference in crystal unit cells arises from a slight reorientation of the two proteins in the better diffracting crystal, such that we had to perform molecular replacement (in two steps, one protein at a time) and extensive re-refinement [MOLREP (Vagin & Teplyakov, 2000) and REFMAC5 (Murshudov et al., 1997) were used] in the crystal form studied here. This protocol was completed by interactive model rebuilding in Coot (Emsley & Cowtan, 2004). The overall quality of the reference model is presented in Table 1.
Diffraction data were measured to a resolution of 1.00 Å, practically limited by the beam wavelength and the minimum crystal-to-detector distance (as displayed in Table 1, despite the high resolution of the data the Rsym is still fairly low in the outer resolution shell). The structure was solved by molecular replacement using the program Phaser; the search model (edited from PDB entry 2d9i ) is an NMR structure consisting of 20 conformers and has a low identity (19%) to the crystallized protein. All 20 conformers of the search model were edited and loops with a large r.m.s.d. between conformers were deleted. The CHAINSAW program was used to edit nonconserved residue side chains, removing all atoms after Cγ. Phaser produced a reasonable molecular-replacement solution for both copies (with a log-likelihood gain of 51.4).
The quality of the positioned search model was assessed using rigid-body refinement (one rigid body per protein copy), leading to an Rwork of 53%. Extensive positional and ADP refinement were not able to reduce the Rwork below 46%. Both these refinements were performed using REFMAC5.
The reference model for comparisons was obtained by submitting the molecular-replacement model to the classic ARP/wARP package (v.6.1.1, using the currently distributed control system) for model rebuilding. This was complemented by iterative refinement in REFMAC5 (Murshudov et al., 1997) and interactive rebuilding in Coot (Emsley & Cowtan, 2004). Anisotropic atomic displacement and multiple conformations were added at the final steps of refinement. The reference model (Table 1) needs improvement before it can be considered `final'; however, it is good enough to be used as a reference in assessment of the results of automatic model rebuilding.
The quality of the search model was assessed a posteriori by computing the r.m.s. deviation between the search model and the reference structure. A least-squares superposition using Cα atoms resulted in an r.m.s.d. of 1.14 Å for 58 out of 82 total residues that were conserved in the alignment.
Automatic molecular replacement by BALBES was systematically attempted on all structures released by the PDB between 22 September and 9 October 2006; the search models were structures released before 21 September 2006. When BALBES produced a molecular-replacement solution according to internal criteria and this model could be related to the deposited structure in the PDB (taking into account potential origin shifts), this example case was added to the set of test structures. A few structures for which we did not manage to reproduce the R factor of the deposited model were excluded. Finally, because flex-wARP currently only handles proteins, we removed data sets for which more than 10% of the ordered diffracting matter was not made of proteins. These two filtering criteria removed a total of 33 data sets out of 162.
The test set thus contains structures that could be solved using a non-identical search model that existed in the PDB prior to the deposition of the test case. For each such case, we store the diffraction data deposited by the authors, the final model deposited by the authors to be used as a reference, the sequence of the protein(s) extracted from the final model and the search model positioned at the right place of the asymmetric unit by BALBES.
The final set comprises 129 structures and is fairly representative of the content of the PDB, with the resolution of the diffraction data spanning from 1.05 to 3.1 Å (median at 2.1 Å), a solvent content ranging between 32 and 72% (median at 50%) and Wilson B factors in the range 4.1–76.8 Å2 (median at 19.3 Å2).
For each data set the deposited model was moved to the same origin as the output of the molecular replacement and a likelihood-weighted electron-density map was computed in REFMAC5 without any positional or ADP refinement (only scaling was applied). The model structure and the electron-density map were then used to assess the quality of the models produced by flex-wARP.
All three protocols (as described in §§2.2, 2.3 and 2.4) were tested systematically. Iteration in flex-wARP was stopped when more than 95% of the residues were built (including side chains) or once 50 steps of model building had been completed, regardless of the total execution time or the number of refinement/update steps. The default protocol, in which the positioned search model is iteratively edited following refinement without removing restraints, was used as a baseline when evaluating the results of the other two protocols (Fig. 5).
For each data set from the BALBES test set, the following information was gathered before starting the model rebuilding, reflecting the statistics available to the crystallographer just after the end of the molecular-replacement search.
To train the estimator, we used as a target function the correlation of the final flex-wARP map to the reference map. For each tested model, an analysis of variance (with a χ2 test) was used to assess which parameters were relevant and which were of little predictive value (comparing the tested model with all the models obtained by systematically dropping one term). Different models were then compared using the Akaike Information Criterium that is designed to reward goodness of fit but also includes a penalty that is an increasing function of the number of degrees of freedom (the goal being to avoid overfitting). All models tested were linear models where the input parameters were either one of the metrics listed above, some power of these parameters or a product of different parameters. The modelling was performed using the R environment (R Development Core Team) and the MASS package (Venables & Ripley, 2002).
ARP/wARP and the new flex-wARP control system are well suited for rebuilding and completing models obtained by molecular replacement. Whilst limited conclusions can be drawn from the two specific examples we present, our medium-scale study based on data of a broader range of quality and resolution provides knowledge that is both more reliable and can be applied to new data sets.
The primary result of this sampling experiment is that ARP/wARP is fairly resilient to poor molecular-replacement solution at high and medium resolution (extending to around 2.5 Å), whilst it can also be useful at lower resolution provided that the molecular-replacement solution is close enough to the true structure. Going further in the analysis, we showed that the protocol that uses the model produced by molecular replacement, including the attached chemical restraints, is the most successful one; however, it is still advisable to blindly test the three proposed protocols to be sure to get the most out of ARP/wARP. As also illustrated in Fig. 7, in almost one out of five cases (19%) it is worth trying the nondefault protocols. To facilitate systematic tests of all available protocols, we are planning to provide a web service to the community.
Finally, we were able to use a simple linear model approximation to express the relative importance of the resolution of the data and the quality of the molecular-replacement solution in obtaining a complete model and a good-quality map using flex-wARP. Overcoming the limitations of the proposed estimator might be achieved by incorporating new training data set as the BALBES development team provides the result of more test rounds and moving away from a linear model to a sigmoid response model or some other supervised learning technique. Despite the scatter visible in Fig. 6, this estimator is useful when `extreme' values are obtained: obtaining an estimate value under 0.6 is a good reason to spend more time obtaining better phasing information (through a better search model or combining molecular replacement with experimental phasing), while obtaining a value greater than 0.8 should almost guarantee success in automated model building.
SXC thanks NWO/CW (VENI 700.55.405), SXC, MBJ, AP and VSL thank the NIH (grant No. R01), JL thanks NWO/CW (VENI 700.53.407), GNM and FL thank the Wellcome Trust (grant No. 064405/Z/01/A) and AAV thanks BBSRC (grant No. 1 RO1 GM069758-03) for funding. This work was performed under the coordination of the EU BIOXHIT FW6 Integrated Project (grant NO. LSHG-CT-2003-503420).
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