research papers\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

IUCrJ
Volume 3| Part 4| July 2016| Pages 259-270
ISSN: 2052-2525

Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

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aInstitute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, England, bResearch Complex at Harwell, STFC Rutherford Appleton Laboratory, Didcot OX11 0FA, England, and cScience and Technology Facilities Council, Daresbury Laboratory, Warrington WA4 4AD, England
*Correspondence e-mail: drigden@liv.ac.uk

Edited by Z.-J. Liu, Chinese Academy of Sciences, China (Received 10 February 2016; accepted 18 May 2016; online 15 June 2016)

For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based) structure prediction. Such models can be used in structure solution by molecular replacement (MR) where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions (`decoys'), is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue–residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing.

1. Introduction

Molecular replacement (MR) is the most common technique for deriving the lost phase information of the unknown target structure in X-ray crystallography. MR places a structurally similar protein in the unit cell of the unknown target to best reproduce the diffraction data. The correct placement of the similar structure provides the basis for the initial phase calculation of the target. The obtained phasing information and the measured diffraction intensities allow the initial calculation of the electron-density map (Blow & Rossmann, 1961[Blow, D. M. & Rossmann, M. G. (1961). Acta Cryst. 14, 1195-1202.]). However, homologues of the target structure do not always exist or may be too structurally distinct. On the other hand, experimental alternatives to MR such as anomalous dispersion (Matthews, 1966[Matthews, B. W. (1966). Acta Cryst. 20, 230-239.]; Hendrickson et al., 1985[Hendrickson, W. A., Smith, J. L. & Sheriff, S. (1985). Methods Enzymol. 115, 41-55.]; Wang, 1985[Wang, B.-C. (1985). Methods Enzymol. 115, 90-112.]) or isomorphous replacement (Green et al., 1954[Green, D. W., Ingram, V. M. & Perutz, M. F. (1954). Proc. R. Soc. Lond. A Math. Phys. Sci. 225, 287-307.]; Perutz, 1956[Perutz, M. F. (1956). Acta Cryst. 9, 867-873.]; Blow & Rossmann, 1961[Blow, D. M. & Rossmann, M. G. (1961). Acta Cryst. 14, 1195-1202.]) can be time-consuming or difficult to implement in certain cases. These considerations have driven recent developments in computational crystallo­graphy to derive and trial search models from unconventional sources. Such sources include ideal secondary-structure elements or structural motifs (Rodríguez et al., 2009[Rodríguez, D. D., Grosse, C., Himmel, S., González, C., de Ilarduya, I. M., Becker, S., Sheldrick, G. M. & Usón, I. (2009). Nat. Methods, 6, 651-653.]), libraries of tertiary structural cores derived from mining the Protein Data Bank (PDB; Sammito et al., 2013[Sammito, M., Millán, C., Rodríguez, D. D., de Ilarduya, I. M., Meindl, K., De Marino, I., Petrillo, G., Buey, R. M., de Pereda, J. M., Zeth, K., Sheldrick, G. M. & Usón, I. (2013). Nat. Methods, 10, 1099-1101.]) and ab initio protein structure predictions (`decoys'; Qian et al., 2007[Qian, B., Raman, S., Das, R., Bradley, P., McCoy, A. J., Read, R. J. & Baker, D. (2007). Nature (London), 450, 259-264.]; Rigden et al., 2008[Rigden, D. J., Keegan, R. M. & Winn, M. D. (2008). Acta Cryst. D64, 1288-1291.]; Das & Baker, 2009[Das, R. & Baker, D. (2009). Acta Cryst. D65, 169-175.]; Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]; Keegan et al., 2015[Keegan, R. M., Bibby, J., Thomas, J., Xu, D., Zhang, Y., Mayans, O., Winn, M. D. & Rigden, D. J. (2015). Acta Cryst. D71, 338-343.]; Rämisch et al., 2015[Rämisch, S., Lizatović, R. & André, I. (2015). Acta Cryst. D71, 606-614.]; Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.]). Clearly, the success of approaches based on ab initio protein modelling will depend sensitively on the quality of the structure predictions available.

In recent years, a step change in the accuracy of residue–residue contact predictions (Giraud et al., 1999[Giraud, B. G., Heumann, J. M. & Lapedes, A. S. (1999). Phys. Rev. E, 59, 4983-4991.]; Miller & Eisenberg, 2008[Miller, C. S. & Eisenberg, D. (2008). Bioinformatics, 24, 1575-1582.]; Weigt et al., 2009[Weigt, M., White, R. A., Szurmant, H., Hoch, J. A. & Hwa, T. (2009). Proc. Natl Acad. Sci. USA, 106, 67-72.]; Burger & van Nimwegen, 2010[Burger, L. & van Nimwegen, E. (2010). PLoS Comput. Biol. 6, e1000633.]), based on sequence information alone, has enabled striking advances to be made in structural bioinformatics, including in ab initio modelling. Although evolutionary covariance analysis for contact prediction is a research area with a long history (Levitt & Warshel, 1975[Levitt, M. & Warshel, A. (1975). Nature (London), 253, 694-698.]; Vendruscolo et al., 1997[Vendruscolo, M., Kussell, E. & Domany, E. (1997). Fold. Des. 2, 295-306.]), only recently has the prediction of such contacts become sufficiently accurate to guide ab initio structure prediction successfully (Supplementary Fig. S1; Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]; Kosciolek & Jones, 2014[Kosciolek, T. & Jones, D. T. (2014). PLoS One, 9, e92197.]; Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]; Adhikari et al., 2015[Adhikari, B., Bhattacharya, D., Cao, R. & Cheng, J. (2015). Proteins, 83, 1436-1449.]; Ovchinnikov et al., 2015[Ovchinnikov, S., Kinch, L., Park, H., Liao, Y., Pei, J., Kim, D. E., Kamisetty, H., Grishin, N. V. & Baker, D. (2015). eLife, 4, e09248.]). The basic rationale behind the prediction of residue–residue contacts rests on the existence of strong evolutionary constraints, reflected in the covariation of contacting residues, to maintain functionally important conformations. Such evolutionary constraints can be detected at a sequence level, but thousands of homologous protein sequences are required to detect them. The great challenge of separating direct and indirect residue–residue contacts (direct, A–B and B–C; indirect, A–C) was recently overcome through the use of cooperative (`global') statistical probability models. These approaches not only treat contact pairs independently, but analyse their dependence on each other, thereby increasing the signal-to-noise ratio in the predicted list of contacts or `contact map' (Giraud et al., 1999[Giraud, B. G., Heumann, J. M. & Lapedes, A. S. (1999). Phys. Rev. E, 59, 4983-4991.]; Miller & Eisenberg, 2008[Miller, C. S. & Eisenberg, D. (2008). Bioinformatics, 24, 1575-1582.]; Weigt et al., 2009[Weigt, M., White, R. A., Szurmant, H., Hoch, J. A. & Hwa, T. (2009). Proc. Natl Acad. Sci. USA, 106, 67-72.]; Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]). Therefore, contacts with the strongest signal, indicated by the highest global statistical scores, are most likely to represent the true residue interactions in a protein conformation (Marks et al., 2012[Marks, D. S., Hopf, T. A. & Sander, C. (2012). Nat. Biotechnol. 30, 1072-1080.]).

Since the successful separation of direct and indirect contacts, various evolutionary covariance-analysis applications have been developed to increase the accuracy and speed of contact predictions (Balakrishnan et al., 2011[Balakrishnan, S., Kamisetty, H., Carbonell, J. G., Lee, S.-I. & Langmead, C. J. (2011). Proteins, 79, 1061-1078.]; Jeong & Kim, 2012[Jeong, C.-S. & Kim, D. (2012). Protein Eng. Des. Sel. 25, 705-713.]; Jones et al., 2012[Jones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2012). Bioinformatics, 28, 184-190.]; Ekeberg et al., 2013[Ekeberg, M., Lövkvist, C., Lan, Y., Weigt, M. & Aurell, E. (2013). Phys. Rev. E, 87, 012707.], 2014[Ekeberg, M., Hartonen, T. & Aurell, E. (2014). J. Comput. Phys. 276, 341-356.]; Kamisetty et al., 2013[Kamisetty, H., Ovchinnikov, S. & Baker, D. (2013). Proc. Natl Acad. Sci. USA, 110, 15674-15679.]; Wang & Xu, 2013[Wang, Z. & Xu, J. (2013). Bioinformatics, 29, i266-i273.]; Feinauer et al., 2014[Feinauer, C., Skwark, M. J., Pagnani, A. & Aurell, E. (2014). PLoS Comput. Biol. 10, e1003847.]; Kaján et al., 2014[Kaján, L., Hopf, T. A., Kalaš, M., Marks, D. S. & Rost, B. (2014). BMC Bioinformatics, 15, 85.]; Schneider & Brock, 2014[Schneider, M. & Brock, O. (2014). PLoS One, 9, e108438.]; Seemayer et al., 2014[Seemayer, S., Gruber, M. & Söding, J. (2014). Bioinformatics, 30, 3128-3130.]; Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]). Broadly, these applications can be divided into three categories depending on the cooperative statistical model implemented to derive evolutionary covariance amongst multiple homologous sequences. The first category employs a pseudo-likelihood maximization model and can be found in applications such as plmDCA (Ekeberg et al., 2013[Ekeberg, M., Lövkvist, C., Lan, Y., Weigt, M. & Aurell, E. (2013). Phys. Rev. E, 87, 012707.]; Kamisetty et al., 2013[Kamisetty, H., Ovchinnikov, S. & Baker, D. (2013). Proc. Natl Acad. Sci. USA, 110, 15674-15679.]), GREMLIN (Kamisetty et al., 2013[Kamisetty, H., Ovchinnikov, S. & Baker, D. (2013). Proc. Natl Acad. Sci. USA, 110, 15674-15679.]) or CCMpred (Seemayer et al., 2014[Seemayer, S., Gruber, M. & Söding, J. (2014). Bioinformatics, 30, 3128-3130.]). The second and third categories include sparse covariance matrix inversion models such as PSICOV (Jones et al., 2012[Jones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2012). Bioinformatics, 28, 184-190.]) or mean-field direct coupling analysis models such as EVFold-mfDCA (Kaján et al., 2014[Kaján, L., Hopf, T. A., Kalaš, M., Marks, D. S. & Rost, B. (2014). BMC Bioinformatics, 15, 85.]), respectively. Although these methods differ in accuracy and speed, a recent study (Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]) revealed a high similarity of around 90% in top-ranked contacts amongst those three categories. To capture the full spectrum of top-ranked contacts and produce the best possible contact map, metapredictors such as PconsC2 (Skwark et al., 2013[Skwark, M. J., Abdel-Rehim, A. & Elofsson, A. (2013). Bioinformatics, 29, 1815-1816.], 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]) or MetaPSICOV (Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]) combine individual contact predictions across two or three of these categories. Additionally, an ongoing aim of prediction tools is to achieve successful detection of evolutionary covariance from homologous sequence sets of reduced diversity and number. Any decrease in the number of homologous sequences required would make covariance analysis applicable to smaller protein families.

One of the principal applications of predicted contact maps is to predict structures for large protein families (Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]; Kosciolek & Jones, 2014[Kosciolek, T. & Jones, D. T. (2014). PLoS One, 9, e92197.]; Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]; Adhikari et al., 2015[Adhikari, B., Bhattacharya, D., Cao, R. & Cheng, J. (2015). Proteins, 83, 1436-1449.]; Ovchinnikov et al., 2015[Ovchinnikov, S., Kinch, L., Park, H., Liao, Y., Pei, J., Kim, D. E., Kamisetty, H., Grishin, N. V. & Baker, D. (2015). eLife, 4, e09248.]). Without contact information, accurate homology-independent fold predictions for globular proteins in Rosetta are limited to chain lengths of up to ∼130 residues (Kinch et al., 2011[Kinch, L., Yong Shi, S., Cong, Q., Cheng, H., Liao, Y. & Grishin, N. V. (2011). Proteins, 79, 59-73.]; He et al., 2013[He, Z., Alazmi, M., Zhang, J. & Xu, D. (2013). PLoS One, 8, e74006.]; Tai et al., 2014[Tai, C.-H., Bai, H., Taylor, T. J. & Lee, B. (2014). Proteins, 82, 57-83.]). Several covariance analysis tools, such as GREMLIN (Ovchinnikov et al., 2015[Ovchinnikov, S., Kinch, L., Park, H., Liao, Y., Pei, J., Kim, D. E., Kamisetty, H., Grishin, N. V. & Baker, D. (2015). eLife, 4, e09248.]), PconsC2 (Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]; Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]), MetaPSICOV (Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]; Kosciolek & Jones, 2015[Kosciolek, T. & Jones, D. T. (2015). Proteins, doi:10.1002/prot.24863.]) and EVFold (Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]), reported accurate fold predictions for much larger globular proteins, illustrating how the use of contact predictions can greatly expand the capabilities of ab initio folding protocols.

The availability of improved tertiary-structure predictions from contact-assisted fragment-assembly ab initio modelling naturally enhances the prospects for their use in MR. Broadly speaking, two approaches have been reported. The first entails highly CPU-intensive modelling to produce an overall fold prediction that is sufficiently accurate to serve as a search model in the same way as a crystal structure or homology model would conventionally be deployed (Qian et al., 2007[Qian, B., Raman, S., Das, R., Bradley, P., McCoy, A. J., Read, R. J. & Baker, D. (2007). Nature (London), 450, 259-264.]; Das & Baker, 2009[Das, R. & Baker, D. (2009). Acta Cryst. D65, 169-175.]). Alternatively, more cheaply obtained, coarse-grained models can be clustered into search-model ensembles and, recognizing their limited accuracy, treated to truncation to attempt to isolate sufficiently accurate core regions (Rigden et al., 2008[Rigden, D. J., Keegan, R. M. & Winn, M. D. (2008). Acta Cryst. D64, 1288-1291.]; Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). AMPLE (ab initio modelling of proteins for molecular replacement; Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]) is a pipeline that implements the latter strategy and is available in the CCP4 software suite (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]). AMPLE overcomes the absence of suitable crystal structures or homology models through a cluster-and-truncate approach that processes computationally cheap and minimally refined Rosetta (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]) or QUARK (Keegan et al., 2015[Keegan, R. M., Bibby, J., Thomas, J., Xu, D., Zhang, Y., Mayans, O., Winn, M. D. & Rigden, D. J. (2015). Acta Cryst. D71, 338-343.]) ab initio decoys into search models. In brief, 1000 ab initio decoys are clustered based on their structural similarity, after which the decoys in the largest resulting cluster (containing a maximum of 200 decoys) are truncated at 20 different intervals. Truncation is rationally guided by inter-decoy structural variance within the cluster (Qian et al., 2007[Qian, B., Raman, S., Das, R., Bradley, P., McCoy, A. J., Read, R. J. & Baker, D. (2007). Nature (London), 450, 259-264.]; Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). The truncated decoys are then subclustered under three different Cα r.m.s.d. radii (1, 2 and 3 Å), whereby a maximum of 30 decoys within the cluster, those closest to the cluster centroid, are selected and combined into an ensemble. Lastly, each ensemble search model undergoes three different side-chain treatments: polyalanine (all side chains are truncated at their Cβ atom), reliable side chains (only side chains are kept that are usually well modelled; Shapovalov & Dunbrack, 2007[Shapovalov, M. V. & Dunbrack, R. L. Jr (2007). Proteins, 66, 279-303.]) and all-atom (all side chains are kept). Up to 120 ensemble search models can be obtained per target through this cluster-and-truncate approach, but this number strongly depends on the structural similarity of the initial decoys and the similarity of the decoys after truncation. Each ensemble search model is processed using MrBUMP (Keegan & Winn, 2008[Keegan, R. M. & Winn, M. D. (2008). Acta Cryst. D64, 119-124.]), which in turn uses Phaser (McCoy et al., 2007[McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658-674.]) and/or MOLREP (Vagin & Teplyakov, 1997[Vagin, A. & Teplyakov, A. (1997). J. Appl. Cryst. 30, 1022-1025.], 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) for MR, SHELXE (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]; Thorn & Sheldrick, 2013[Thorn, A. & Sheldrick, G. M. (2013). Acta Cryst. D69, 2251-2256.]) for main-chain tracing and ARP/wARP (Cohen et al., 2008[Cohen, S. X., Ben Jelloul, M., Long, F., Vagin, A., Knipscheer, P., Lebbink, J., Sixma, T. K., Lamzin, V. S., Murshudov, G. N. & Perrakis, A. (2008). Acta Cryst. D64, 49-60.]) or Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) for automatic rebuilding of the SHELXE trace.

In an initial study of 295 small globular proteins with fewer than 120 residues and resolution better than 2.2 Å, 43% of the targets were solved successfully (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). However, the application of ab initio modelling to MR, and therefore the success of AMPLE, is greatly limited by the size and fold class of the protein target. These limitations arise at the initial stage during decoy prediction, where successful fold predictions of protein structures without homologues of known structure are currently limited to a chain length of ∼130 residues (Kinch et al., 2011[Kinch, L., Yong Shi, S., Cong, Q., Cheng, H., Liao, Y. & Grishin, N. V. (2011). Proteins, 79, 59-73.]; He et al., 2013[He, Z., Alazmi, M., Zhang, J. & Xu, D. (2013). PLoS One, 8, e74006.]; Tai et al., 2014[Tai, C.-H., Bai, H., Taylor, T. J. & Lee, B. (2014). Proteins, 82, 57-83.]). AMPLE has been shown to succeed with protein targets greater than 150 amino acids in size, but these cases were not comparable in the nature of the target and/or the methodology employed: the successes were achieved with either anisometric folds (e.g. coiled coils), ensemble search models derived from distant structural homologues or NMR structures (Bibby et al., 2013[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2013). Acta Cryst. D69, 2194-2201.]; Bruhn et al., 2014[Bruhn, J. F., Barnett, K. C., Bibby, J., Thomas, J. M. H., Keegan, R. M., Rigden, D. J., Bornholdt, Z. A. & Saphire, E. O. (2014). J. Virol. 88, 758-762.]; Hotta et al., 2014[Hotta, K., Keegan, R. M., Ranganathan, S., Fang, M., Bibby, J., Winn, M. D., Sato, M., Lian, M., Watanabe, K., Rigden, D. J. & Kim, C.-Y. (2014). Angew. Chem. Int. Ed. 53, 824-828.]; Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.]). Thus, the largest globular protein target previously solved with AMPLE using ab initio models is 120 amino acids in length (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]), although it was noted in that work that success rates had not declined to zero at this size threshold. In addition to the issue of protein size, the success rate of AMPLE strongly depends on the fold architecture, as reflected in the widely varying success rates of all-α (80%), mixed αβ (including α/β and α+β folds; 37%) and all-β protein targets (2%) in the original test set of 295 small globular proteins. In sum, both the size and the fold of the target can limit ab initio folding protocols and thus the success rate of AMPLE.

Ab initio modelling of proteins without exploiting information from known folds is a longstanding challenge in the field of computational structural biology and success currently strongly depends on the chain length and fold architecture of the target protein. Recent successful advances in the derivation of direct residue–residue contacts from large multiple sequence alignments have greatly increased the accuracy of ab initio structure predictions, especially for larger and all-β protein targets, which are the greatest challenges for ab initio-based MR approaches. Here, we set out to explore the impact of the improved contact-guided decoys on the success rate of MR. For this, we use our automated pipeline AMPLE, as its cluster-and-truncate approach has proven to be highly successful in the downstream processing of ab initio decoys for MR (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). We report that contact-guided decoys allow the successful solution of targets that were previously unsolvable using the AMPLE method. In addition, we report that combining independently obtained contact maps further improves decoy quality, which in turn extends the tractable MR target range to β-rich proteins.

2. Methods

2.1. Data set

A test set of 21 globular protein targets was used throughout. They were manually selected to include a range of chain lengths, fold architectures, X-ray diffraction data resolutions and divergent sequence counts in a multiple sequence alignment. The test set covered the three fold classes (α-helical, mixed αβ and β-sheet) and each target was grouped based on its secondary-structure content as defined by DSSP (Kabsch & Sander, 1983[Kabsch, W. & Sander, C. (1983). Biopolymers, 22, 2577-2637.]; Joosten et al., 2011[Joosten, R. P., te Beek, T. A. H., Krieger, E., Hekkelman, M. L., Hooft, R. W. W., Schneider, R., Sander, C. & Vriend, G. (2011). Nucleic Acids Res. 39, D411-D419.]; Supplementary Table S1). The chain length of the sequences ranged from 62 to 221 residues and each crystal structure contained one molecule in the asymmetric unit. The resolutions of the crystal structures ranged from 1.0 to 2.3 Å. The FASTA sequences of each target, as provided in the PDB entry (Rose et al., 2015[Rose, P. W., Prlić, A., Bi, C., Bluhm, W. F., Christie, C. H., Dutta, S., Green, R. K., Goodsell, D. S., Westbrook, J. D., Woo, J., Young, J., Zardecki, C., Berman, H. M., Bourne, P. E. & Burley, S. K. (2015). Nucleic Acids Res. 43, D345-D356.]), were modelled, rather than the sequence that was visibly present in the crystallographic model. A number of divergent (`effective') sequences (Neff) available for a target of greater than 100 is considered to be the minimum requirement for accurate covariance-based contact predictions (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]). The formula Neff = [\sum[(1/n_1) + (1/n_2) + \ldots + (1/n_i)]] (Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]) defines Neff as the sum of fractional weights of n sequences in i clusters in a multiple sequence alignment (MSA). To calculate this parameter for our targets, each target sequence formed the basis of an MSA which was obtained from a database search with HHblits v.2.0.15 (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nat. Methods, 9, 173-175.]). Two sequence-search iterations were performed with an E-value cutoff of 10−3 against the nonredundant UniProt20 database v.2013.03 (The UniProt Consortium, 2015[The UniProt Consortium (2015). Nucleic Acids Res. 43, D204-D212.]). All sequences in each resulting alignment were then clustered using CD-HIT v.4.6.3 (Li et al., 2001[Li, W., Jaroszewski, L. & Godzik, A. (2001). Bioinformatics, 17, 282-283.], 2002[Li, W., Jaroszewski, L. & Godzik, A. (2002). Bioinformatics, 18, 77-82.]; Fu et al., 2012[Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. (2012). Bioinformatics, 28, 3150-3152.]) at 62% sequence identity (Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]) and Neff was calculated.

2.2. Evolutionary covariance analysis

One contact map was predicted for each of the 21 targets using the fully automated metapredictor PconsC2 (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]). In summary, MSAs were generated with Jackhmmer (Johnson et al., 2010[Johnson, L. S., Eddy, S. R. & Portugaly, E. (2010). BMC Bioinformatics, 11, 431.]) against the UniRef100 database and with HHblits v.2.0.15 (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nat. Methods, 9, 173-175.]) against the non­redundant UniProt20 database v.2013.03 (The UniProt Consortium, 2015[The UniProt Consortium (2015). Nucleic Acids Res. 43, D204-D212.]) at E-value cutoffs of 10−40, 10−10, 10−4 and 1. Each MSA was then analysed with PSICOV (Jones et al., 2012[Jones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2012). Bioinformatics, 28, 184-190.]) and plmDCA (Ekeberg et al., 2013[Ekeberg, M., Lövkvist, C., Lan, Y., Weigt, M. & Aurell, E. (2013). Phys. Rev. E, 87, 012707.], 2014[Ekeberg, M., Hartonen, T. & Aurell, E. (2014). J. Comput. Phys. 276, 341-356.]) to produce 16 individual sets of contact predictions. All 16 predictions, combined with a secondary-structure prediction, solvent-accessibility information and a sequence profile, were then provided to a deep-learning algorithm (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]) to identify protein-like contact patterns. The latter produced a final contact map for each target sequence.

An additional contact map for β-structure-containing targets was predicted using CCMpred (Seemayer et al., 2014[Seemayer, S., Gruber, M. & Söding, J. (2014). Bioinformatics, 30, 3128-3130.]) and reduced to β-sheet contact pairs using the CCMpred-specific filtering protocol bbcontacts (Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]). Each MSA for CCMpred contact predictions was obtained using HHblits v.2.0.15 (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nat. Methods, 9, 173-175.]). This entailed two sequence-search iterations with an E-value cutoff of 10−3 against the nonredundant UniProt20 database v.2013.03 (The UniProt Consortium, 2015[The UniProt Consortium (2015). Nucleic Acids Res. 43, D204-D212.]) and filtering to 90% sequence identity using HHfilter v.2.0.15 (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nat. Methods, 9, 173-175.]) to reduce sequence redundancy in the MSA. Besides the contact matrix as input, bbcontacts requires a secondary-structure prediction and a factor describing the range of predicted contacts in the MSA. The latter was shown to depend on the sequence count in the MSA (N) and the target chain length (L). Thus, the factor describing this MSA-specific diversity was calculated using the equation η = (N/L)1/2 (Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]). The secondary structure for each sequence was predicted using the addss.pl (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nat. Methods, 9, 173-175.]) script distributed with HH-suite v.2.0.16 (Söding, 2005[Söding, J. (2005). Bioinformatics, 21, 951-960.]). Hereafter, the term bbcontacts will be used to describe the full process from the target sequence to the filtered β-strand contact map. At no point do contact-prediction algorithms use structural information from structurally characterized proteins.

2.3. Conversion of contact maps to contact restraints

For all targets, the predicted contact maps from PconsC2 were converted to Rosetta (Rohl et al., 2004[Rohl, C. A., Strauss, C. E. M., Misura, K. M. S. & Baker, D. (2004). Methods Enzymol. 383, 66-93.]) restraints to guide ab initio folding of the target sequences. The FADE energy function was used to introduce a restraint in the folding protocol of Rosetta. As described in PconsFold (Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]), a restraint was satisfied during folding if the participating Cβ atoms (Cα in the case of glycine) were within 9 Å of one another. If a pre-defined contact restraint was satisfied, a smoothed `squared-well' bonus was added to the internal energy scoring function of Rosetta during folding. The shape of this function therefore rewards conformations that place residues within 9 Å of each other, but has no influence on the energy outside this range. Thus, a false-positive prediction between two positions that are in fact distant in the target structure will not lead to an undesirable long-distance attraction between the two residues. As defined by Michel et al. (2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]), the `squared-well' bonus (parameter wd in FADE) was set to −15.00. Adopting the same benchmarked approach as Michel et al. (2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]), only the top L ranked contacts (based on confidence scores, with L again representing target length) from each PconsC2 contact map were selected and converted to Rosetta restraints.

For β-containing targets, an alternative selection of predicted contacts, hereafter called PconsC2+bbcontacts, was made by a novel combination of PconsC2 and bbcontacts predictions, as follows. Firstly, inter-strand predictions composed of only one or two contacts were removed from the bbcontacts contact list owing to their high false-positive rate (Jessica Andreani, personal communication). For all present contact pairs between residues i and j and any neighbouring contacts (i.e. i, j ± 1; i, j ± 2; i ± 1, j; i ± 2, j) in the top-L PconsC2 contact list the `squared-well' bonus was doubled from −15.00 to −30.00, which proved to be the most effective after several options were tried (unpublished data). In addition, all contact pairs solely present in the filtered bbcontacts contact map were added to the modified PconsC2 and bbcontacts contact list with a `squared-well' bonus of −15.00. It is worth noting that the added bbcontacts contacts were also present in the full PconsC2 contact map, although they were not within the top-L cutoff. This approach allowed a strengthening of the weight on β-strand contacts during ab initio structure prediction. After uniting the two predictions in this way no further length-based cutoff was applied, so that the PconsC2+bbcontacts restraint list fed to Rosetta for β-containing proteins might be longer than the simple PconsC2 list.

The final contact-prediction lists were compared with the corresponding crystal structure contacts to determine their accuracy. For this, all pairs of Cβ atoms (Cα in the case of glycine) within 9 Å of one another in the crystal structure were considered as reference contacts. Predictions were assigned as true or false positives according to whether they were in the list of reference contacts or not. The precision or positive predictive value (PPV) for the each restraint list was then determined using the formula PPV = (true positives)/(true positives + false positives).

2.4. Ab initio structure prediction of decoys

Fragments were picked for unassisted Rosetta modelling with secondary-structure prediction from PSIPRED (McGuffin et al., 2000[McGuffin, L. J., Bryson, K. & Jones, D. T. (2000). Bioinformatics, 16, 404-405.]) and for contact-assisted decoys with the secondary-structure prediction obtained during evolutionary covariance analysis. Homologous structures were excluded using the nohoms flag to make all experiments equivalent to predictions of unknown folds. Protein decoys were generated using the AbinitioRelax folding protocol of Rosetta in v.2015wk05 (Rohl et al., 2004[Rohl, C. A., Strauss, C. E. M., Misura, K. M. S. & Baker, D. (2004). Methods Enzymol. 383, 66-93.]). As recommended in the Rosetta documentation, special modelling parameters included the helix and loop atom-refinement flags abinitio::rsd_wt_helix and abinitio::rsd_wt_loop with a reweight factor of 0.5. The two flags abinitio:relax and relax::fast were set during ab initio modelling to obtain an all-atom refinement using the Rosetta full-atom force field. For each target in the data set, the structure folds were predicted under two different restraint conditions: without any residue–residue contact restraints and with PconsC2-only contact restraints. Targets containing β-folds were additionally modelled with a third restraint condition: PconsC2+bbcontacts contact restraints derived as described above. bbcontacts-only contact restraints were not treated as a separate condition owing to the low count of predicted contacts. A total of 1000 decoys were modelled for each target under each of the three different restraint conditions. Decoy quality was assessed based on the template-modelling score (TM-score; Zhang & Skolnick, 2005[Zhang, Y. & Skolnick, J. (2005). Nucleic Acids Res. 33, 2302-2309.]), a measure of fold similarity between two structures with identical sequences, in this case a decoy and the corresponding crystal structure. TM-scores range from 0 to 1, with a TM-score above 0.5 usually indicating a correct fold prediction.

2.5. Molecular replacement

The three sets of ab initio decoys for each target were subjected to SCWRL4 side-chain remodelling (Canutescu et al., 2003[Canutescu, A. A., Shelenkov, A. A. & Dunbrack, R. L. (2003). Protein Sci. 12, 2001-2014.]; Krivov et al., 2009[Krivov, G. G., Shapovalov, M. V. & Dunbrack, R. L. Jr (2009). Proteins, 77, 778-795.]). Afterwards, all three sets of decoys for each target were run in the automated MR pipeline AMPLE v.1.0 using default parameters, with the exception of the number of clusters to trial, which was changed from one to three (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). The associated structure-factor amplitudes for the crystal structure of each target were retrieved from the PDB. The correct placement of search models by Phaser was assessed using the recently developed residue-independent overlap (RIO) score (Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.]). In short, the RIO score assesses the in-sequence and out-of-sequence register overlap of the placed search-model residues (fragments of at least three residues) with the corresponding crystal structure. To be considered a success, MR using AMPLE was required to give a SHELXE correlation coefficient (CC) of ≥25.00 and an average chain length (ACL) of ≥10.00 (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]; Thorn & Sheldrick, 2013[Thorn, A. & Sheldrick, G. M. (2013). Acta Cryst. D69, 2251-2256.]; Keegan et al., 2015[Keegan, R. M., Bibby, J., Thomas, J., Xu, D., Zhang, Y., Mayans, O., Winn, M. D. & Rigden, D. J. (2015). Acta Cryst. D71, 338-343.]). Additionally, structure rebuilding of the SHELXE chain traces was attempted using both ARP/wARP (Cohen et al., 2008[Cohen, S. X., Ben Jelloul, M., Long, F., Vagin, A., Knipscheer, P., Lebbink, J., Sixma, T. K., Lamzin, V. S., Murshudov, G. N. & Perrakis, A. (2008). Acta Cryst. D64, 49-60.]) and Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) and an Rfree value of ≤0.45 from either method was required.

3. Results

3.1. Both general and β-strand-specific residue–residue contact maps improve ab initio protein structure predictions

The initial part of this study evaluates the use of contact restraints for improving ab initio protein structure prediction. For each protein target in the data set, 1000 decoys were predicted using Rosetta, either unassisted or with restraints deriving from PconsC2 alone or from our novel fusion of PconsC2 and bbcontacts predictions. Since AMPLE by default processes decoys from the largest cluster to create search models, structure quality was primarily assessed for these structure predictions alone. However, we also report overall improvements for all decoys (Table 1[link]).

Table 1
Restraint-guided ab initio modelling improves model quality

Median template-modelling scores for ab initio decoys found in the largest cluster (values for all decoys are shown in parentheses) predicted for threefold classes using three different types of residue–residue constraint settings.

Fold classification Rosetta PconsC2-only PconsC2+bbcontacts
All-α 0.377 (0.298) 0.609 (0.531)
Mixed αβ 0.314 (0.252) 0.537 (0.433) 0.565 (0.441)
All-β 0.323 (0.247) 0.467 (0.374) 0.471 (0.381)
Mixed αβ + all-β 0.320 (0.249) 0.506 (0.397) 0.522 (0.403)

A high degree of sequence diversity is a prerequisite for the identification of residue covariance in MSAs. The range of effective sequences in the MSAs of target sequences ranged from 272 to 1831. Typically, higher numbers of effective sequences correlate with more accurate contact predictions (Jones et al., 2012[Jones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2012). Bioinformatics, 28, 184-190.]; Kamisetty et al., 2013[Kamisetty, H., Ovchinnikov, S. & Baker, D. (2013). Proc. Natl Acad. Sci. USA, 110, 15674-15679.]; Ekeberg et al., 2014[Ekeberg, M., Hartonen, T. & Aurell, E. (2014). J. Comput. Phys. 276, 341-356.]; Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]; Ma et al., 2015[Ma, J., Wang, S., Wang, Z. & Xu, J. (2015). Bioinformatics, 31, 3506-3513.]). Here, similar results were observed, as illustrated in Supplementary Fig. S2. Considering the three fold classes, more accurate predictions were obtained for β-structure-containing proteins (median PPVβ = 0.940; median PPVαβ = 0.909) compared with all-α targets (median PPVα = 0.655). In plain language, for β-structure-containing proteins over 90% of the intramolecular contacts predicted by evolutionary covariance methods are indeed present in the crystal structure. Although untested in this study, a higher accuracy for β-sheet-containing proteins is achieved owing to the regular pattern of contact pairs that is easily detectable in a contact map (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]; Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]). The deep-learning procedure used during the final step of PconsC2 filters these contact pairs better, therefore increasing the overall accuracy of the prediction.

As expected (Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]; Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]), the inclusion of PconsC2-predicted contact information substantially improved the quality of structure predictions. A simple Rosetta run without contact information yielded a largest cluster median TM-score of 0.342 for all 21 protein targets, compared with 0.542 for PconsC2-only decoys (Fig. 1[link]a). 20 of 21 targets were modelled better, with median TM-score improvements ranging from 0.035 to 0.429. A single target, PDB entry 2qyj, the unassisted Rosetta models for which were already of exceptionally high quality (median TM-score of largest cluster structure predictions of 0.865), was modelled slightly worse (0.780) when contact information was included.

[Figure 1]
Figure 1
Residue–residue contact restraints improve ab initio model quality. (a) Median template-modelling scores (TM-scores) for Rosetta decoys plotted against median TM-scores for PconsC2-only coupling-guided decoys. (b) Median TM-score for PconsC2-only coupling-guided decoys plotted against median TM-scores for PconsC2+bbcontacts decoys (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]; Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]). Median TM-scores derived from decoys found in the largest cluster. The symbol shapes correspond to the three different fold classes: all-α (circles), all-β (triangles) and mixed αβ (squares).

For 13 β-strand-containing proteins in the data set we developed a novel approach of combining the top-L predicted PconsC2 (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]) contacts with the filtered β-sheet-specific bbcontacts (Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]) contacts. This procedure resulted in the upweighting of some contacts already present in the PconsC2 list and the addition of others. The number of contacts affected in each category is shown in Fig. 2[link]. At least 80% of upweighted contact restraints (present in the final contact lists of PconsC2 and bbcontacts predictions) proved to be true positives (Fig. 2[link]a). The average PPV of upweighted contacts was 95% (Fig. 2[link]a). The quality of the added contacts was generally lower (Fig. 2[link]b), but nine of the 13 targets had a PPV of at least 50% (Fig. 2[link]b). Thus, at the cost of the inclusion of some false-positive contacts, our approach generally provides extra valuable information for the folding process.

[Figure 2]
Figure 2
Effects of upweighting and addition of β-sheet-specific contacts on contact accuracy and decoy quality. Number of (a) upweighted and (b) added bbcontacts β-sheet-specific contact restraints for 13 β-sheet-containing (seven all-β, triangles; six mixed αβ, squares) targets plotted against their corresponding positive predictive value (PPV). The colour fill of each point corresponds to the resulting difference in median TM-scores between the largest cluster decoys from PconsC2-only and PconsC2+bbcontacts decoy sets (positive values favour the latter).

Models based on PconsC2+bbcontacts contacts were again somewhat improved compared with those built using the PconsC2 contacts: the median TM-scores for the two model sets were 0.522 and 0.506, respectively (Fig. 1[link]b). Model quality improved for nine targets, of which five showed improvements in median TM-score of at least 0.02 (Table 2[link]). Model quality deteriorated for four targets, but for three of these the difference was very small: less than 0.02 (Table 2[link]).

Table 2
Summary of ab initio structure-prediction results of β-structure-containing targets

All data shown are for PconsC2+bbcontacts (PconsC2-only) guided decoys.

          TM-score
Fold classification PDB code No. of effective sequences No. of contacts PPV Top-cluster decoys 1000 decoys
Mixed αβ 1aba 1037 92 (87) 0.787 (0.782) 0.584 (0.546) 0.507 (0.496)
1chd 852 222 (203) 0.924 (0.931) 0.626 (0.634) 0.501 (0.528)
1e0s 1831 184 (174) 0.691 (0.713) 0.520 (0.495) 0.362 (0.353)
1eaz 1060 136 (125) 0.928 (0.944) 0.581 (0.547) 0.512 (0.460)
1lo7 1026 146 (141) 0.980 (0.986) 0.535 (0.528) 0.453 (0.443)
1tjx 1189 178 (159) 0.857 (0.887) 0.425 (0.427) 0.354 (0.358)
All-β 1bdo 940 91 (80) 0.913 (0.963) 0.477 (0.490) 0.379 (0.402)
1kjl 272 183 (146) 0.704 (0.727) 0.385 (0.367) 0.313 (0.293)
1npu 943 136 (117) 0.835 (0.940) 0.389 (0.438) 0.322 (0.331)
1pnc 887 111 (99) 0.830 (0.889) 0.478 (0.470) 0.375 (0.354)
2nuz 1048 70 (62) 0.901 (0.952) 0.650 (0.622) 0.540 (0.498)
3w56 949 146 (131) 0.896 (0.906) 0.444 (0.440) 0.363 (0.353)
4u3h 1226 109 (100) 0.911 (0.950) 0.563 (0.461) 0.440 (0.446)

3.2. Contact-guided ab initio models extend the tractable target range of AMPLE

With a demonstrable and significant improvement in decoy quality evident from the use of predicted contact restraints, the ability of AMPLE to solve the 21 protein targets using contact-guided decoys was then tested. For all targets, two sets of decoys were trialled deriving from ab initio structure prediction with no contact-prediction restraints or with PconsC2-only restraints. For β-strand-containing targets a third decoy set was created using PconsC2+bbcontacts-derived restraints. Structure solution of each target was attempted with these sets of structure predictions using default AMPLE methods. Successful MR was detected as previously by the ability of SHELXE main-chain tracing and density modification run on the MR placement to reach a correlation coefficient (CC) of ≥25 with a mean traced chain length of ≥10 residues (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]; Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]; Thorn & Sheldrick, 2013[Thorn, A. & Sheldrick, G. M. (2013). Acta Cryst. D69, 2251-2256.]; Keegan et al., 2015[Keegan, R. M., Bibby, J., Thomas, J., Xu, D., Zhang, Y., Mayans, O., Winn, M. D. & Rigden, D. J. (2015). Acta Cryst. D71, 338-343.]; Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.]). As previously (Keegan et al., 2015[Keegan, R. M., Bibby, J., Thomas, J., Xu, D., Zhang, Y., Mayans, O., Winn, M. D. & Rigden, D. J. (2015). Acta Cryst. D71, 338-343.]; Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.]), we further required that an Rfree value of ≤0.45 could be achieved after ARP/wARP (Cohen et al., 2008[Cohen, S. X., Ben Jelloul, M., Long, F., Vagin, A., Knipscheer, P., Lebbink, J., Sixma, T. K., Lamzin, V. S., Murshudov, G. N. & Perrakis, A. (2008). Acta Cryst. D64, 49-60.]) or Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) automatic rebuilding of the resulting SHELXE chain traces.

Based on these stringent success criteria, the default algorithm of AMPLE achieved eight structure solutions for decoys predicted without contact restraints (Fig. 3[link], blue). Six out of eight all-α, one mixed αβ and one all-β target were solved with chain lengths up to 213 residues. Success using ab initio models has not been previously reported for such large globular protein targets (Table 3[link]), but these findings recapitulate the fold-class preferences observed previously: AMPLE works well for all-α targets but less so for mixed αβ and particularly all-β proteins (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). Previously, the strong performance on all-α targets has been at least partly attributed to the greater accuracy of Rosetta modelling of those proteins (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]), but these results make a second important contribution more explicit. Most all-α targets were solved despite the overall accuracy of their models being poor (Fig. 3[link]). This suggests that their success in MR, nevertheless, lies with the superior ability of SHELXE to autotrace helices compared with other secondary structures (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]). The β-containing targets solved here were the mixed α/β-fold bacteriophage T4 glutaredoxin (PDB entry 1aba) and the all-β biotinyl domain of acetyl-coenzyme A carboxylase (PDB entry 1bdo), both with chain lengths of less than 90 residues (Table 3[link]).

Table 3
Summary of molecular-replacement solutions of 21 protein targets

The total number of ensemble search models derived from ab initio decoys from the three largest clusters is provided in parentheses after the individual number of successful search models.

        No. of successful (total) search models
Fold classification PDB code Resolution (Å) Target chain length Rosetta PconsC2-only PconsC2+bbcontacts
All-α 1kw4 1.75 89 137 (393) 101 (468)
1bkr 1.10 109 21 (105) 13 (459)
4cl9 1.40 127 1 (210) 1 (408)
1a6m 1.00 151 1 (102) 4 (327)
2qyj 2.05 166 378 (501) 329 (453)
4w97 1.60 200 6 (114) 3 (399)
1hh8 1.80 213 3 (66) 0 (297)
1tlv 1.95 221 0 (18) 2 (399)
Mixed αβ 1aba 1.45 87 4 (312) 58 (429) 93 (411)
1eaz 1.40 125 0 (135) 0 (345) 28 (327)
1lo7 1.50 141 0 (120) 3 (327) 3 (333)
1tjx 1.04 159 0 (27) 0 (165) 0 (150)
1e0s 2.28 174 0 (15) 0 (195) 0 (207)
1chd 1.75 203 0 (12) 0 (279) 0 (225)
All-β 2nuz 1.85 62 0 (393) 76 (444) 183 (453)
1bdo 1.80 80 27 (343) 16 (381) 19 (372)
1pnc 1.60 99 0 (126) 0 (300) 0 (297)
4u3h 1.98 100 0 (273) 14 (357) 1 (372)
1npu 2.00 117 0 (111) 0 (210) 0 (180)
3w56 1.60 131 0 (129) 0 (123) 0 (150)
1kjl 1.40 146 0 (63) 0 (174) 0 (201)
[Figure 3]
Figure 3
Contact restraint-guided ab initio models extend the tractable target range of AMPLE. Molecular-replacement (MR) success mapped against target chain length and median template-modelling score (TM-score). The point shape corresponds to the fold class of the target: all-α (circles), all-β (triangles) and mixed αβ (squares). The point colour indicates successful structure solutions for the contact constraints used: none (blue), PconsC2-only (red) and PconsC2+bbcontacts (gold). Points for successful solutions were considered in the order of Rosetta, PconsC2-only and PconsC2+bbcontacts decoys. In cases of unsuccessful molecular-replacement attempts (empty symbols), TM-scores for the largest clusters of PconsC2+bbcontacts decoys are shown. Median TM-scores for each point correspond to the largest decoy cluster (compared with the crystal structure), leading to a structure solution (cluster indices given next to each point for targets that were not solved with the largest cluster). The dashed grey line highlights the tested target chain-length limit of AMPLE (120 residues) for globular proteins (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). Cartoon representations of crystal structures of five different targets exemplify the diversity of structure solutions (PDB identifiers are provided next to each crystal structure). α-Helices are shown in red, β-sheets in yellow and loops in green.

When predicted contact information from PconsC2 was used in the modelling, the resultant structure predictions from the largest cluster solved an additional two all-β structure solutions: the α-spectrin SH3 domain (PDB entry 2nuz) and the FN3con domain (PDB entry 4u3h) (Fig. 3[link], red). Although these targets do not exceed the previously benchmarked chain-size limit of 120 residues (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]), it is worth noting that three out of the five all-β-containing proteins with chain lengths of less than 120 residues were solved. This strongly indicates that the previously low success rate of AMPLE of ∼2% for all-β targets in this size range (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]) is improved by using contact information. PconsC2+bbcontacts decoys achieved all of the structure solutions using PconsC2-only decoys. Additionally and most notably, PconsC2+bbcontacts decoys led to the structure solution of the mixed α+β PH domain of the human TAPP1 protein (PDB entry 1eaz; Fig. 3[link], gold). The structure solution of 1eaz uniquely using PconsC2+bbcontacts restraints highlights the importance of the fusion of contact maps developed here. In total, the largest cluster decoys modelled with PconsC2 and bbcontacts restraints solved 11 out of 21 targets.

By default, the AMPLE algorithm processes ab initio models solely from the largest cluster. When trialling ensemble search models based on decoys from the three largest clusters, an additional three structure solutions were obtained (Fig. 3[link]). The successful solution of haem-bound oxymyoglobin (PDB entry 1a6m) was achieved with Rosetta decoys from the third cluster. Notably, ab initio modelling of this target was performed without its large bound haem group, yet structure solution was achieved. The second target, the inactive LicT PRD domain from Bacillus subtilis (PDB entry 1tlv), was solved with PconsC2-only decoys from the third cluster. This all-α target with a chain length of 221 residues is, to our knowledge, the largest globular protein to be solved using search models derived from ab initio protein structure modelling. As mentioned above, all-α targets such as this benefit from the powerful helix tracing in SHELXE (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]), as do the programs of the ARCIMBOLDO suite (Rodríguez et al., 2009[Rodríguez, D. D., Grosse, C., Himmel, S., González, C., de Ilarduya, I. M., Becker, S., Sheldrick, G. M. & Usón, I. (2009). Nat. Methods, 6, 651-653.]), which can also solve large all-α protein structures (Fourati et al., 2014[Fourati, Z., Roy, B., Millan, C., Coureux, P.-D., Kervestin, S., van Tilbeurgh, H., He, F., Usón, I., Jacobson, A. & Graille, M. (2014). J. Mol. Biol. 426, 3689-3702.]). Lastly, PconsC2-only and PconsC2+bbcontacts decoys derived from second largest clusters yielded search models that solved the 4-hydroxy­benzoyl CoA thioesterase domain structure (PDB entry 1lo7). Particularly notable about this solution is the topology of the search models. Although this mixed α+β target contains a number of helices, the best structure solutions (based on SHELXE CC scores) were obtained from search models containing the accurately modelled, central, four-stranded β-sheet (Fig. 4[link]). This accurate modelling, which is required for successful MR, was only achieved with the guidance of contact restraints. In total, the addition of these three structure solutions results in 14 out of 21 structure solutions for PconsC2+bbcontacts decoys compared with nine for simple Rosetta decoys.

[Figure 4]
Figure 4
Contact restraints improve the search-model quality of β-strand-containing targets. Structural superposition of the (a) Rosetta (Cα r.m.s.d. 2.814 Å; ensemble contains two structures), (b) PconsC2-only (Cα r.m.s.d. 1.748 Å; 30 members) and (c) PconsC2+bbcontacts (Cα r.m.s.d. 1.760 Å; 15 members) search-model ensembles for 4-hydroxybenzoyl CoA thioesterase (PDB entry 1lo7). Examples are the highest scoring search models based on SHELXE CC score, with only (b) and (c) leading to successful structure solutions. Search models are shown as tubes and crystal structures as cartoons. (a) and (c) are 50% of the target sequence, while (b) is 55%. The colour scale illustrates the pairwise Cα r.m.s.d. between each search-model ensemble (represented by its first member) and the crystal structure, with blue representing the minimum Cα r.m.s.d. and red the maximum. Unaligned residues are coloured grey.

Although the stringent criteria of MR success used here did not indicate a successful structure solution for target 1e0s, the beneficial effect of including joint PconsC2+bbcontacts contact predictions was evident in the search-model placement as assessed by RIO scores (Fig. 5[link]). For the top PconsC2-only search model, 40% (12 residues) of the search-model residues were correctly superimposed, albeit out of register (blue) on the target structure (Phaser TFZ = 4.7, Phaser LLG = 16). For the top PconsC2+bbcontacts search model, 77% (30 residues) of the search model were superimposed in an in-register fashion (Phaser TFZ = 5.3, Phaser LLG = 17) (Fig. 5[link]). For the latter, expert manual intervention might allow structure determination, but in this case the correct solution was not prominent in the list of MR placements. Nevertheless, it is clear that even when overall structure solution was not automatically achieved the PconsC2+bbcontacts model provided better results which might be recoverable as successes in the future as MR and post-MR software improves still further.

[Figure 5]
Figure 5
PconsC2+bbcontacts contact-derived models give a more accurate MR placement for PDB entry 1e0s which, although not solved automatically, might yield to expert manual intervention. Top Phaser solutions of 1e0s based on RIO scores for (a) PconsC2-only (RIO score 12) and (b) PconsC2+bbcontacts (RIO score 30) search models for target 1e0s. Search-model colour coding indicates useful superposition of residues by in-sequence (green) or out-of-sequence register (blue) residues as well as misplaced (red) residues. The addition of bbcontacts restraints produced a more accurate model with correctly placed β-strands that was placed correctly. Both structures are shown in cartoon representation with the crystal structure shown as a transparent cartoon. Unaligned reference crystal structure residues are coloured grey.

Within the range explored, the success of structure solution did not appear to depend significantly on the resolution of the available crystallographic data (Supplementary Fig. S3). Successful targets ranged in resolution from 1.00 to 2.05 Å (mean ± standard deviation of 1.62 ± 0.32 Å), while unsuccessful targets spanned 1.04–2.28 Å (1.67 ± 0.40 Å). The solvent content of the protein crystals appeared to have a modest impact on MR success. Targets with successful structure solutions ranged from 36.0 to 55.3% (mean ± SD of 46.1 ± 5.2%) solvent content compared with 25.8–48.0% (39.1 ± 8.1%) for unsuccessful targets.

Given that the inclusion of predicted contact information is a significant change to the modelling protocol, we re-examined the performance and importance of the key features of the operation of AMPLE. A detailed analysis of the characteristics of the successful search models is provided in the Supporting Information; only a summary is provided here. AMPLE uses well established clustering of decoys (Simons et al., 1997[Simons, K. T., Kooperberg, C., Huang, E. & Baker, D. (1997). J. Mol. Biol. 268, 209-225.]) to pick out those likely to be the most accurate. This continues to be effective here, as picking the largest clusters selects better than average decoys from the sets available (Supplementary Fig. S4) and there is a good correlation between the largest cluster size and the median TM-score of the decoys in that cluster (Supplementary Fig. S5). However, the size of the largest cluster does not correlate well with the total number of successful search models (Supplementary Fig. S6). AMPLE also relies on rational, variance-based truncation to trim ensembles down to more accurate core structures, with the size range 15–40 residues found to be most successful (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). Here, the truncation is further validated (Fig. 6[link]) and a similar mapping of success onto search-model size is observed (Supplementary Fig. S7). Fig. 6[link] further illustrates the overall positive impact of contacts on accuracy: note the larger number of low-r.m.s.d. ensembles on the right of Fig. 6[link](b) compared with Fig. 6[link](a). However, Fig. 6[link] also illustrates that targets that are already well modelled by simple Rosetta and successful in MR (blue points on the right in Fig. 6[link]a) can be modelled slightly worse when contact information is included (somewhat raised r.m.s.d.s in Fig. 6[link]b), presumably owing to the influence of false-positive contact predictions. Previously, we have found that sampling across three subclustering radii and three modes of side-chain treatment were both required for solution of the largest possible number of targets (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]). This remains largely the case in the current exercise, as unique solutions were obtained for each of the sub­clustering radii (Supplementary Table S1). Polyalanine side-chain search models were the most successful, but a single target, PDB entry 1eaz, was only solved using one of the alternative treatments (Supplementary Table S1).

[Figure 6]
Figure 6
Variance-based truncation remains an effective way to derive successful search models from higher quality contact restraint-assisted ab initio decoys. The percentage of sequence in the search model is mapped against the root-mean-square deviation (r.m.s.d.) over all Cα atoms of the first representative of each search-model ensemble derived from the largest cluster against the native structure. Successful structure solutions of individual search models are highlighted in blue and unsuccessful solutions in red. Progressively darker shades of either colour correspond to increasing numbers of overlapping points. Progressive truncation is shown for (a) Rosetta decoys and (b) PconsC2+bbcontacts decoys (or PconsC2-only decoys for all-α targets).

4. Discussion

The recently emerged ability to predict contacting residues from large protein sequence alignments is one of the most exciting developments in structural bioinformatics for many years. The key statistical breakthrough allowing the disentangling of predicted direct contacts (Giraud et al., 1999[Giraud, B. G., Heumann, J. M. & Lapedes, A. S. (1999). Phys. Rev. E, 59, 4983-4991.]; Miller & Eisenberg, 2008[Miller, C. S. & Eisenberg, D. (2008). Bioinformatics, 24, 1575-1582.]; Weigt et al., 2009[Weigt, M., White, R. A., Szurmant, H., Hoch, J. A. & Hwa, T. (2009). Proc. Natl Acad. Sci. USA, 106, 67-72.]; Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]), i.e. neighbouring amino acids from pairs of residues whose identities covary indirectly, has been followed by a wave of papers not only dealing with the accuracy of predictions but also considering the manifold applications of the information. Predicted contact information is of immediate benefit to crystallographers in many ways that are yet to be fully appreciated, including parsing of domains for structural analysis (Rigden, 2002[Rigden, D. J. (2002). Protein Eng. 15, 65-77.]; Sadowski, 2013[Sadowski, M. I. (2013). Proteins, 81, 253-260.]) and interpretation of crystal structure composition (Nicoludis et al., 2015[Nicoludis, J. M., Lau, S.-Y., Schärfe, C. P. I., Marks, D. S., Weihofen, W. A. & Gaudet, R. (2015). Structure, 23, 2087-2098.]). Here, we considered how the better protein ab initio models that can be produced by exploiting information can serve as a source of improved search models for MR. We use the MR pipeline AMPLE as a convenient and effective tool for the analysis.

Challenged by the lesser success of AMPLE with β-structure-containing proteins (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]), and motivated by the accuracy improvements in ab initio fold predictions through contact restraints (Marks et al., 2011[Marks, D. S., Colwell, L. J., Sheridan, R., Hopf, T. A., Pagnani, A., Zecchina, R. & Sander, C. (2011). PLoS One, 6, e28766.]; Michel et al., 2014[Michel, M., Hayat, S., Skwark, M. J., Sander, C., Marks, D. S. & Elofsson, A. (2014). Bioinformatics, 30, i482-i488.]; Jones et al., 2015[Jones, D. T., Singh, T., Kosciolek, T. & Tetchner, S. (2015). Bioinformatics, 31, 999-1006.]; Ovchinnikov et al., 2015[Ovchinnikov, S., Kinch, L., Park, H., Liao, Y., Pei, J., Kim, D. E., Kamisetty, H., Grishin, N. V. & Baker, D. (2015). eLife, 4, e09248.]), we developed a new approach for combining predicted contact-restraint lists from PconsC2 (Skwark et al., 2014[Skwark, M. J., Raimondi, D., Michel, M. & Elofsson, A. (2014). PLoS Comput. Biol. 10, e1003889.]) and bbcontacts (Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]) to elevate the ab initio modelling accuracy of β-structure protein targets. Structure predictions guided by the resulting PconsC2+bbcontacts contact restraints improved the decoy quality for nine out of 13 β-structure-containing protein targets. Our approach, which involved both selective upweighting of and addition to the PconsC2 set, based on the specialist β-sheet predictions, may well be of more general use to the protein-modelling community. The value of these contact-guided ab initio models for structure solution by MR of targets treated as novel folds is demonstrated. Nine of the 21 targets in the data set were solved using the AMPLE algorithm to process unassisted Rosetta structure predictions. This number rose to 14 using contact-guided modelling. The 100% success rate for all-α targets is highly encouraging and, along with the comparable MR pipeline ARCIMBOLDO (Rodríguez et al., 2009[Rodríguez, D. D., Grosse, C., Himmel, S., González, C., de Ilarduya, I. M., Becker, S., Sheldrick, G. M. & Usón, I. (2009). Nat. Methods, 6, 651-653.]), graphically illustrates the power of the α-helical tracing implemented in SHELXE (Sheldrick, 2010[Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.]) and the relative tractability of α-rich targets to unconventional MR. More β-rich, α-poor targets are harder for both AMPLE and ARCIMBOLDO, so our demonstrable advances with these targets, leveraging the value of contact restraints during ab initio modelling, are exciting. The fusion of top-ranked PconsC2 and bbcontacts contacts developed as part of this study proved to be a key part in one successful structure solution, further highlighting the importance of the approach.

The size of the targets solved is another notable feature of this work. We originally suggested (Bibby et al., 2012[Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622-1631.]) that all-α protein targets larger than the 120-residue threshold then tested could be suitable for the cluster-and-truncate approach of AMPLE. Here, we demonstrate this to be true, with unassisted decoys leading to the solution of a 213-residue protein and contact-assisted models leading to the successful solution of a 221-residue chain. To our knowledge, these are the largest targets to be solved with search models derived from ab initio structure decoys.

The availability of reliable contact restraints to aid MR with ab initio models clearly widens the range of targets for which AMPLE is a viable option for structure solution. The accuracy of contact predictions is directly related to the number of protein sequences deposited in sequence databases such as UniProt (The UniProt Consortium, 2015[The UniProt Consortium (2015). Nucleic Acids Res. 43, D204-D212.]), and thus will benefit from the continuous growth of those databases. Notably, this manuscript focused solely on globular proteins; yet the AMPLE algorithm is equally well suited to coiled-coil and transmembrane proteins (Thomas et al., 2015[Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198-206.] and unpublished data). Specific contact predictors for the latter are available (Wang et al., 2011[Wang, X.-F., Chen, Z., Wang, C., Yan, R.-X., Zhang, Z. & Song, J. (2011). PLoS One, 6, e26767.]; Hopf et al., 2012[Hopf, T. A., Colwell, L. J., Sheridan, R., Rost, B., Sander, C. & Marks, D. S. (2012). Cell, 149, 1607-1621.]; Yang et al., 2013[Yang, J., Jang, R., Zhang, Y. & Shen, H.-B. (2013). Bioinformatics, 29, 2579-2587.]; Zhang et al., 2016[Zhang, H., Huang, Q., Bei, Z., Wei, Y. & Floudas, C. A. (2016). Proteins, 84, 332-348.]) and future research will explore their application to MR using AMPLE. In conclusion, the current and future broadening of the target range tractable by AMPLE through the use of evolutionary restraints during ab initio modelling highlights the value of the software as an effective alternative to experimental phasing approaches in X-ray crystallography.

In summary, we confirm here that predicted contacts can significantly improve ab initio model quality in a way that directly impacts on structure solution by MR. Our novel mode of uniting general and β-structure-specific contact predictions brings further tangible model improvements to the particularly difficult β-rich protein targets. All of these methodo­logical advances have immediate benefits for crystallographers facing targets with novel or divergent folds which cannot be addressed by conventional MR. AMPLE proves to be an efficient framework for rendering these contact-assisted decoys into search-model ensembles, with truncation and extensive sampling remaining key to success. Future inevitable expansion of sequence databases and predictable improvements in contact-prediction software will undoubtedly extend the reach of MR with ab initio models still further.

Supporting information


Footnotes

Present address: Department of Biology, Unversität Konstanz, 78457 Konstanz, Germany.

Acknowledgements

This work was supported by BBSRC grant BB/L009544/1 `CCP4 Grant Renewal 2014–2019: Question-driven crystallo­graphic data collection and advanced structure solution'. FS and JT are supported by BBSRC DTP PhD scholarships.

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IUCrJ
Volume 3| Part 4| July 2016| Pages 259-270
ISSN: 2052-2525