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CRYSTALLOGRAPHY
ISSN: 1600-5767

The MORPHEUS protein crystallization screen

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aMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, England
*Correspondence e-mail: fgorrec@mrc-lmb.cam.ac.uk

(Received 31 July 2009; accepted 13 October 2009; online 7 November 2009)

A 96-condition initial screen for protein crystallization, called MORPHEUS, has been developed at the MRC Laboratory of Molecular Biology, Cambridge, England (MRC-LMB). The concept integrates several innovative approaches, such as chemically compatible mixes of potential ligands, new buffer systems and precipitant mixes that also act as cryoprotectants. Instead of gathering a set of crystallization conditions that have already been successful, a selection of molecules frequently observed in the Protein Data Bank (PDB) to co-crystallize with proteins has been made. These have been put together in mixes of similar chemical behaviour and structure, and combined with buffers and precipitant mixes that were also derived from PDB searches, to build the screen de novo. Observations made at the MRC-LMB and many practical aspects were also taken into account when formulating the screen. The resulting screen is easy to use, comprehensive yet small, and has already yielded a list of crystallization hits using both known and novel samples. As an indicator of success, the screen has now become one of the standard screens used routinely at the MRC-LMB when searching initial crystallization conditions for biological macromolecules.

1. Introduction

Structure determination of biological macromolecules has been tremendously successful over recent years. The Protein Data Bank (PDB, https://www.pdb.org; Berman et al., 2000[Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N. & Bourne, P. E. (2000). Nucleic Acids Res. 28, 235-242.]) now holds nearly 60 000 coordinate sets. Approximately 80% of those have been determined by X-ray crystallography, and the method, since its first application to biological macromolecules more than 50 years ago (Kendrew et al., 1958[Kendrew, J. C., Bodo, G., Dintzi, H. M., Parrish, R. G., Wyckoff, H. & Phillips, D. C. (1958). Nature (London), 181, 662-666.]; Perutz et al., 1960[Perutz, M. F., Rossmann, M. G., Af, C., Muirhead, H., Will, G. & North, A. C. (1960). Nature (London), 185, 416-422.]), has continued to improve. Recently, the atomic structure of the complete 70S ribosome was determined using X-ray crystallography (Selmer et al., 2006[Selmer, M., Dunham, C. M., Murphy, F. V. T., Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R. & Ramakrishnan, V. (2006). Science, 313, 1935-1942.]). Given the obvious successes, one might be forgiven for assuming that the basis of the method, the crystallization of a protein, DNA or RNA and their complexes, must be an easy process. In fact, crystallization is now rate limiting and a typical project trying to elucidate the structure of a biological macromolecule of interest will spend most time trying to obtain a sample of biological interest that can be crystallized (Chayen & Saridakis, 2008[Chayen, N. E. & Saridakis, E. (2008). Nat. Methods, 5, 147-153.]). The underlying problem is that at the time of the crystallization experiment the structure of the molecule is not known and hence a rational approach cannot be taken.

To circumvent this problem, crystallization screens are utilized which try to sample the vast number of possible variables in a manageable and efficient way, either systematically or randomly (McPherson, 2004[McPherson, A. (2004). Methods, 34, 254-265.]). Development of an effective search strategy depends on determining how parameter variations influence crystal formation and crystal quality (Kingston et al., 1994[Kingston, R. L., Baker, H. M. & Baker, E. N. (1994). Acta Cryst. D50, 429-440.]). The protein itself can be considered as the main variable (Dale et al., 2003[Dale, G. E., Oefner, C. & D'Arcy, A. (2003). J. Struct. Biol. 142, 88-97.]). However, the correct composition of the initial crystallization screen is necessary, although by no means sufficient, for success.

Nowadays, vapour diffusion with 50–200 nl drops is the most widespread crystallization technique and many different commercial screening kits are available to initiate experiments (Berry et al., 2006[Berry, I. M., Dym, O., Esnouf, R. M., Harlos, K., Meged, R., Perrakis, A., Sussman, J. L., Walter, T. S., Wilson, J. & Messerschmidt, A. (2006). Acta Cryst. D62, 1137-1149.]). Many screens are systematic variations of the concentrations or chemical nature of the components and others employ so-called sparse-matrix approaches that are essentially collections of conditions (mixes of reagents used for protein crystallization) that have been found to work previously with other samples (Jancarik & Kim, 1991[Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst. 24, 409-411.]).

The increasing number of structures deposited in the PDB has motivated some statistical analyses of the crystallization conditions employed (Hennessy et al., 2000[Hennessy, D., Buchanan, B., Subramanian, D., Wilkosz, P. A. & Rosenberg, J. M. (2000). Acta Cryst. D56, 817-827.]; Kantardjieff & Rupp, 2004[Kantardjieff, K. A. & Rupp, B. (2004). Bioinformatics, 20, 2162-2168.]), together with attempts to rationalize protein crystallization screens (Zhu et al., 2006[Zhu, D. W., Garneau, A., Mazumdar, M., Zhou, M., Xu, G. J. & Lin, S. X. (2006). J. Struct. Biol. 154, 297-302.]; Newstead et al., 2008[Newstead, S., Ferrandon, S. & Iwata, S. (2008). Protein Sci. 17, 466-472.]). Rationalization has led to screens with a minimal number of conditions in sparse matrices and footprint screens (Brzozowski & Walton, 2001[Brzozowski, A. M. & Walton, J. (2001). J. Appl. Cryst. 34, 97-101.]; Radaev & Sun, 2002[Radaev, S. & Sun, P. D. (2002). J. Appl. Cryst. 35, 674-676.]; Tran et al., 2004[Tran, T. T., Sorel, I. & Lewit-Bentley, A. (2004). Acta Cryst. D60, 1562-1568.]; Newman et al., 2005[Newman, J., Egan, D., Walter, T. S., Meged, R., Berry, I., Ben Jelloul, M., Sussman, J. L., Stuart, D. I. & Perrakis, A. (2005). Acta Cryst. D61, 1426-1431.]). This is logical if overall efficiency is the main goal, such as in structural genomics.

At the MRC Laboratory of Molecular Biology (Cambridge, England), protein samples, DNA–protein complexes and RNA-containing complexes are regularly screened using standard procedures with more than 40 commercial initial screen kits (Stock et al., 2005[Stock, D., Perisic, O. & Löwe, J. (2005). Prog. Biophys. Mol. Biol. 88, 311-327.]) and over 1500 conditions, assembled into pre-filled MRC 96-well crystallization plates. This large number is still not large enough because many samples fail to crystallize or give only a very few hits. Amongst others, this could be due to two main reasons. Firstly, the vast number of possible conditions is under-sampled (which is surely true). Secondly, crystallization can be critically dependent on the component(s) in the screen (St John et al., 2008[St John, F. J., Feng, B. & Pozharski, E. (2008). Acta Cryst. D64, 1222-1227.]) that make proteins behave differently (more stable or rigid, for example). The latter reason is the rationale behind classical additive screening (Cudney et al., 1994[Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y. & McPherson, A. (1994). Acta Cryst. D50, 414-423.]) and a recent development called Silverbullets (McPherson & Cudney, 2006[McPherson, A. & Cudney, B. (2006). J. Struct. Biol. 156, 387-406.]).

Both assumptions were a driving force behind my attempts to formulate the new screen MORPHEUS that could enhance the chances of crystallization. The most important feature of MORPHEUS is the inclusion of mixes containing potential ligands and additives that can promote crystallization through specific interactions. This strategy includes the risk that one component of a mix might have a deleterious effect on crystal growth (or complex association) and thereby mask the positive contribution of another (Larson et al., 2007[Larson, S. B., Day, J. S., Cudney, R. & McPherson, A. (2007). Acta Cryst. D63, 310-318.]). By selecting components that have been seen to be ordered in crystal structures in the PDB, the chances of incorporating molecules playing a positive role should increase.

An extensive search of the PDB was performed and small molecules and ions that bind to biological macromolecules were selected. The molecules are stable, commercially available, have a molecular weight below 250 Da and are easy to handle. Components found abundantly in the PDB are potentially good crystallization agents for two reasons. Firstly, they can be stabilizers. For example, some sugars are well known for their thermodynamic stabilization of macromolecules (Arakawa & Timasheff, 1982[Arakawa, T. & Timasheff, S. N. (1982). Biochemistry, 21, 6536-6544.]). Stabilization can also mean `rigidifying' the protein or the crystal lattice and thus improving diffraction quality. Secondly, ligands can create crystallization variants by changing possible interactions on the molecular surface, hence increasing the chances of obtaining different crystals. From this perspective, small counter-anions like nitrate, phosphate and sulfate, with a multitude of possible binding modes via different spatial arrangements of O atoms, are ideal components. For the same reason, small organic salts with carboxylic acid groups can facilitate crystal growth (McPherson, 2001[McPherson, A. (2001). Protein Sci. 10, 418-422.]). Additional agents found frequently in the PDB include halides that promote different crystal forms (Lim et al., 1998[Lim, K., Nadarajah, A., Forsythe, E. L. & Pusey, M. L. (1998). Acta Cryst. D54, 899-904.]) and can help with crystallographic phase determination (Dauter et al., 2000[Dauter, Z., Dauter, M. & Rajashankar, K. R. (2000). Acta Cryst. D56, 232-237.]). It has been shown that polyethylene glycols (PEGs) tend to form linear binding patterns in clefts on protein surfaces (Hasek, 2006[Hasek, J. (2006). Z. Kristallogr. 23, 613-618.]). Therefore, a selection of six PEGs completes the formulation of MORPHEUS.

MORPHEUS provides 96 original conditions made from innovative mixes of potential ligands that have been found with high frequency in the PDB. Will MORPHEUS, like the Greek god of dreams, take different forms, especially those in the shape of crystals? Here, ideas about the formulations and the results from crystallization experiments using test proteins and novel samples are described, proving the high usability and efficiency of MORPHEUS.

2. Materials and methods

The complete formulation of MORPHEUS is shown in Table 1[link]. Fig. 1[link] is a schematic representation of the screen layout.

Table 1
Formulation of MORPHEUS

PEG MME is polyethylene glycol monomethyl ether. MPD is (RS)-2-methyl-2,4-pentanediol. NPS is a mix containing sodium nitrate, disodium hydrogen phosphate and ammonium sulfate.

Well Mix of precipitants Mix of additives Buffer system
A1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each divalent cation 0.1 M MES/imidazole pH 6.5
A2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each divalent cation 0.1 M MES/imidazole pH 6.5
A3 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each divalent cation 0.1 M MES/imidazole pH 6.5
A4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each divalent cation 0.1 M MES/imidazole pH 6.5
A5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each divalent cation 0.1 M MOPS/HEPES-Na pH 7.5
A6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each divalent cation 0.1 M MOPS/HEPES-Na pH 7.5
A7 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each divalent cation 0.1 M MOPS/HEPES-Na pH 7.5
A8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each divalent cation 0.1 M MOPS/HEPES-Na pH 7.5
A9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each divalent cation 0.1 M bicine/Trizma base pH 8.5
A10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each divalent cation 0.1 M bicine/Trizma base pH 8.5
A11 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each divalent cation 0.1 M bicine/Trizma base pH 8.5
A12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each divalent cation 0.1 M bicine/Trizma base pH 8.5
B1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each halide 0.1 M MES/imidazole pH 6.5
B2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each halide 0.1 M MES/imidazole pH 6.5
B3 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each halide 0.1 M MES/imidazole pH 6.5
B4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each halide 0.1 M MES/imidazole pH 6.5
B5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each halide 0.1 M MOPS/HEPES-Na pH 7.5
B6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each halide 0.1 M MOPS/HEPES-Na pH 7.5
B7 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each halide 0.1 M MOPS/HEPES-Na pH 7.5
B8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each halide 0.1 M MOPS/HEPES-Na pH 7.5
B9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each halide 0.1 M bicine/Trizma base pH 8.5
B10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each halide 0.1 M bicine/Trizma base pH 8.5
B11 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each halide 0.1 M bicine/Trizma base pH 8.5
B12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each halide 0.1 M bicine/Trizma base pH 8.5
C1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each NPS 0.1 M MES/imidazole pH 6.5
C2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each NPS 0.1 M MES/imidazole pH 6.5
C3 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each NPS 0.1 M MES/imidazole pH 6.5
C4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each NPS 0.1 M MES/imidazole pH 6.5
C5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each NPS 0.1 M MOPS/HEPES-Na pH 7.5
C6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each NPS 0.1 M MOPS/HEPES-Na pH 7.5
C7 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each NPS 0.1 M MOPS/HEPES-Na pH 7.5
C8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each NPS 0.1 M MOPS/HEPES-Na pH 7.5
C9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each NPS 0.1 M bicine/Trizma base pH 8.5
C10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each NPS 0.1 M bicine/Trizma base pH 8.5
C11 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each NPS 0.1 M bicine/Trizma base pH 8.5
C12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each NPS 0.1 M bicine/Trizma base pH 8.5
D1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each alcohol 0.1 M MES/imidazole pH 6.5
D2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each alcohol 0.1 M MES/imidazole pH 6.5
D3 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each alcohol 0.1 M MES/imidazole pH 6.5
D4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each alcohol 0.1 M MES/imidazole pH 6.5
D5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each alcohol 0.1 M MOPS/HEPES-Na pH 7.5
D6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each alcohol 0.1 M MOPS/HEPES-Na pH 7.5
D7 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each alcohol 0.1 M MOPS/HEPES-Na pH 7.5
D8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each alcohol 0.1 M MOPS/HEPES-Na pH 7.5
D9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each alcohol 0.1 M bicine/Trizma base pH 8.5
D10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each alcohol 0.1 M bicine/Trizma base pH 8.5
D11 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each alcohol 0.1 M bicine/Trizma base pH 8.5
D12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each alcohol 0.1 M bicine/Trizma base pH 8.5
E1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each ethylene glycol 0.1 M MES/imidazole pH 6.5
E2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each ethylene glycol 0.1 M MES/imidazole pH 6.5
E3 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each ethylene glycol 0.1 M MES/imidazole pH 6.5
E4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each ethylene glycol 0.1 M MES/imidazole pH 6.5
E5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each ethylene glycol 0.1 M MOPS/HEPES-Na pH 7.5
E6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each ethylene glycol 0.1 M MOPS/HEPES-Na pH 7.5
E7 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each ethylene glycol 0.1 M MOPS/HEPES-Na pH 7.5
E8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each ethylene glycol 0.1 M MOPS/HEPES-Na pH 7.5
E9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.03 M of each ethylene glycol 0.1 M bicine/Trizma base pH 8.5
E10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.03 M of each ethylene glycol 0.1 M bicine/Trizma base pH 8.5
E11 10% w/v PEG 4000, 20% v/v glycerol 0.03 M of each ethylene glycol 0.1 M bicine/Trizma base pH 8.5
E12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.03 M of each ethylene glycol 0.1 M bicine/Trizma base pH 8.5
F1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each monosaccharide 0.1 M MES/imidazole pH 6.5
F2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each monosaccharide 0.1 M MES/imidazole pH 6.5
F3 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each monosaccharide 0.1 M MES/imidazole pH 6.5
F4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each monosaccharide 0.1 M MES/imidazole pH 6.5
F5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each monosaccharide 0.1 M MOPS/HEPES-Na pH 7.5
F6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each monosaccharide 0.1 M MOPS/HEPES-Na pH 7.5
F7 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each monosaccharide 0.1 M MOPS/HEPES-Na pH 7.5
F8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each monosaccharide 0.1 M MOPS/HEPES-Na pH 7.5
F9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each monosaccharide 0.1 M bicine/Trizma base pH 8.5
F10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each monosaccharide 0.1 M bicine/Trizma base pH 8.5
F11 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each monosaccharide 0.1 M bicine/Trizma base pH 8.5
F12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each monosaccharide 0.1 M bicine/Trizma base pH 8.5
G1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each carboxylic acid 0.1 M MES/imidazole pH 6.5
G2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each carboxylic acid 0.1 M MES/imidazole pH 6.5
G3 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each carboxylic acid 0.1 M MES/imidazole pH 6.5
G4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each carboxylic acid 0.1 M MES/imidazole pH 6.5
G5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each carboxylic acid 0.1 M MOPS/HEPES-Na pH 7.5
G6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each carboxylic acid 0.1 M MOPS/HEPES-Na pH 7.5
G7 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each carboxylic acid 0.1 M MOPS/HEPES-Na pH 7.5
G8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each carboxylic acid 0.1 M MOPS/HEPES-Na pH 7.5
G9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each carboxylic acid 0.1 M bicine/Trizma base pH 8.5
G10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each carboxylic acid 0.1 M bicine/Trizma base pH 8.5
G11 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each carboxylic acid 0.1 M bicine/Trizma base pH 8.5
G12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each carboxylic acid 0.1 M bicine/Trizma base pH 8.5
H1 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each amino acid 0.1 M MES/imidazole pH 6.5
H2 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each amino acid 0.1 M MES/imidazole pH 6.5
H3 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each amino acid 0.1 M MES/imidazole pH 6.5
H4 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each amino acid 0.1 M MES/imidazole pH 6.5
H5 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each amino acid 0.1 M MOPS/HEPES-Na pH 7.5
H6 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each amino acid 0.1 M MOPS/HEPES-Na pH 7.5
H7 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each amino acid 0.1 M MOPS/HEPES-Na pH 7.5
H8 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each amino acid 0.1 M MOPS/HEPES-Na pH 7.5
H9 10% w/v PEG 20 000, 20% v/v PEG MME 550 0.02 M of each amino acid 0.1 M bicine/Trizma base pH 8.5
H10 10% w/v PEG 8000, 20% v/v ethylene glycol 0.02 M of each amino acid 0.1 M bicine/Trizma base pH 8.5
H11 10% w/v PEG 4000, 20% v/v glycerol 0.02 M of each amino acid 0.1 M bicine/Trizma base pH 8.5
H12 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% v/v MPD 0.02 M of each amino acid 0.1 M bicine/Trizma base pH 8.5
[Figure 1]
Figure 1
MORPHEUS schematic screen layout.

2.1. Selection of PDB-derived ligands

The set of 47 PDB-derived ligands is listed in Table 2[link]. Initially, structures with ligand(s) were tabulated (July, 2008). Data were then filtered with a molecular weight cut-off of 250 Da. The resulting list was filtered again to keep only ligands seen with at least five unrelated protein structures.

Table 2
The 47 PDB-derived ligands selected to formulate MORPHEUS

MPD is (RS)-2-methyl-2,4-pentanediol.

Ligand Residue ID No. of structures
(RS)-Tartaric acid TAR, TLA 113
1,2-(RS)-Propanediol PGR, PGO 41
1,3-Propanediol PDO 7
1,4-Butanediol BU1 11
1,6-Hexanediol HEZ 19
1-Butanol 1BO 7
2-Propanol IPA, IOH 174
Acetate anion ACT, ACY, ACE 1890
Ammonium cation NH4, NH3, NH2 582
Bicine BCN 11
Bromide anion BR 120
Calcium cation CA 3959
Chloride anion CL 2842
Citrate anion FLC, CIT 384
D-Galactose GLA, GAL 86
D-Glucose GLC, BGC 206
Diethylene glycol PEG 209
DL-Alanine ALA, DAL 35
DL-Lysine LYS, DLY 36
DL-Serine SER, DSN 38
D-Mannose MAN, BMA 178
D-Xylose XYP, XYL 33
Ethylene glycol EDO 1081
Fluoride anion F 16
Formic acid FMT 267
Glycerol GOL 2884
Glycine GLY 50
HEPES EPE 201
Imidazole IMD 154
Iodide anion IOD 178
L-Fucose FUC, FUL 62
L-Glutamic acid GLU 28
Magnesium cation MG 3991
MES MES 315
MOPS MPO 21
MPD MRD, MPD 504
N-Acetyl-D-glucosamine NAG 1150
Nitrate anion NO3 156
Oxamic acid OXM 17
Pentaethylene glycol 1PE 91
Phosphate anion PO4, PI, 2HP 1687
Potassium cation K 720
Sodium cation NA 1926
Sulfate anion SO4 5793
Tetraethylene glycol PG4 194
Triethylene glycol PGE 107
Tris TRS 334
     
Total No. of entries   32908

Not included in MORPHEUS because of chemical incompatibility are all phenols, heavy atoms and detergents. Many divalent cations and some carboxylic acids were discarded in later tests because of problems with stability and false positives. Also, there is a limit to the number of ligands (i.e. additives) that can be integrated into 96 conditions. Concentrations must be high because low affinities should be considered (Sauter et al., 1999[Sauter, C., Ng, J. D., Lorber, B., Keith, G., Brion, P., Hosseini, M. W., Lehn, J. M. & Giegé, R. (1999). J. Cryst. Growth, 196, 365-376.]).

2.2. Additive mixes

Thirty-eight of the selected PDB-derived ligands have been grouped into families depending on their chemical nature to form eight additive mixes. For example, one of the additive mixes is composed of n-ethylene glycols (n = 2–5). By grouping the additives based on chemical nature, the possibility of cross-reaction is avoided and stock solutions are stable. When additives were salts with an acid or base form, the salts were selected so that the final pH of the mix was as neutral as possible. A compound-to-protein ratio of 10:1 is commonly adopted for co-crystallization with small molecule ligands (Danley, 2006[Danley, D. E. (2006). Acta Cryst. D62, 569-575.]) and hence the final concentration of each additive in MORPHEUS is 0.02 M minimum, representing ten times the concentration of a 10 kDa protein at 20 mg ml−1. The recipes for preparing the eight MORPHEUS additive mixes can be found in Table 3[link].

Table 3
Recipes for preparing the eight MORPHEUS additive mixes

Stock Composition
Divalent cations 0.3 M magnesium chloride, 0.3 M calcium chloride
Halides 0.3 M sodium fluoride, 0.3 M sodium bromide, 0.3 M sodium iodide
NPS 0.3 M sodium nitrate, 0.3 M disodium hydrogen phosphate, 0.3 M ammonium sulfate
Alcohols 0.2 M 1,6-hexanediol, 0.2 M 1-butanol, 0.2 M (RS)-1,2-propanediol, 0.2 M 2-propanol, 0.2 M 1,4-butanediol, 0.2 M 1,3-propanediol
Ethylene glycols 0.3 M diethyleneglycol, 0.3 M triethyleneglycol, 0.3 M tetraethyleneglycol, 0.3 M pentaethyleneglycol
Monosaccharides 0.2 M D-glucose, 0.2 M D-mannose, 0.2 M D-galactose, 0.2 M L-fucose, 0.2 M D-xylose, 0.2 M N-acetyl-D-glucosamine
Carboxylic acids 0.2 M sodium formate, 0.2 M ammonium acetate, 0.2 M trisodium citrate, 0.2 M sodium potassium L-tartrate, 0.2 M sodium oxamate
Amino acids 0.2 M sodium L-glutamate, 0.2 M DL-alanine, 0.2 M glycine, 0.2 M DL-lysine HCl, 0.2 M DL-serine

2.3. Precipitant mixes

Precipitants can be mixed to have a synergistic effect (Majeed et al., 2003[Majeed, S., Ofek, G., Belachew, A., Huang, C. C., Zhou, T. & Kwong, P. D. (2003). Structure, 11, 1061-1070.]) and/or to provide cryoprotection (Mitchell & Garman, 1994[Mitchell, E. P. & Garman, E. F. (1994). J. Appl. Cryst. 27, 1070-1074.]; McFerrin & Snell, 2002[McFerrin, M. B. & Snell, E. H. (2002). J. Appl. Cryst. 35, 538-545.]). To take advantage of these findings, four precipitant mixes were integrated in the formulation of MORPHEUS. Three of the mixes have been observed to be more successful in the crystallization of MRC-LMB samples than expected from their under-sampling in our initial screens, as described previously. A fourth mix was designed from scratch with components not found in the other three mixes. Principally, the precipitant mixes have been chosen so that the final conditions produce vitrified ice when frozen. It should be noted, however, that the optimal concentration of cryoprotectant is sample dependent and may need optimization later (Chinte et al., 2005[Chinte, U., Shah, B., DeWitt, K., Kirschbaum, K., Pinkerton, A. A. & Schall, C. (2005). J. Appl. Cryst. 38, 412-419.]). Recipes for preparing the four MORPHEUS stock solutions with precipitants can be found in Table 4[link]. The table includes the frequency of similar mixes in our MRC-LMB standard initial screens.

Table 4
Recipes for preparing the four MORPHEUS precipitant mixes

Composition Frequency Reference
20% w/v PEG 20 000, 40% v/v PEG MME 550 35 Cordell et al. (2003[Cordell, S. C., Robinson, E. J. & Löwe, J. (2003). Proc. Natl Acad. Sci. USA, 100, 7889-7894.]); Leonard et al. (2004[Leonard, T. A., Butler, P. J. & Löwe, J. (2004). Mol. Microbiol. 53, 419-432.]); Selmer et al. (2006[Selmer, M., Dunham, C. M., Murphy, F. V. T., Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R. & Ramakrishnan, V. (2006). Science, 313, 1935-1942.])
20% w/v PEG 8000, 40% v/v ethylene glycol 3 Teo et al. (2006[Teo, H., Gill, D. J., Sun, J., Perisic, O., Veprintsev, D. B., Vallis, Y., Emr, S. D. & Williams, R. L. (2006). Cell, 125, 99-111.])
20% w/v PEG 4000, 40% v/v glycerol 12 Low & Löwe (2006[Low, H. H. & Löwe, J. (2006). Nature (London), 444, 766-769.])
25% w/v PEG 3350, 25% w/v PEG 1000, 25% v/v MPD 0 Not published

2.4. Buffer systems

Six of the selected PDB-derived ligands described before have been used to build three buffer systems within a physiological pH range, namely 6.5, 7.5 and 8.5. The common advantage of buffer systems is that no titration with concentrated acid or base is required (Newman, 2004[Newman, J. (2004). Acta Cryst. D60, 610-612.]). Each MORPHEUS buffer system includes an acid and base pair of buffers with similar pKa values. This way, the systems combine the characteristics of two different Good buffers for biological research (Good et al., 1966[Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S. & Singh, R. M. (1966). Biochemistry, 5, 467-477.]).

Recipes for preparing 50 ml of the three MORPHEUS buffer systems can be found in Table 5[link]. Non-titrated stock solutions of the individual buffers (at a concentration of 1 M) were mixed at different ratios for optimization purposes.

Table 5
Recipes for preparing the three MORPHEUS buffer systems at different pH

pH 1 M MES (ml) 1 M imidazole (ml)
6.1 36.0 14.0
6.3 33.5 16.5
6.5 30.6 19.4
6.7 27.5 22.5
6.9 25.0 25.0
pH 1 M MOPS (ml) 1 M HEPES-Na (ml)
7.1 34.5 15.5
7.3 30.0 20.0
7.5 25.9 24.1
7.7 22.1 37.9
7.9 17.7 32.3
pH 1 M bicine (ml) 1 M Trizma base (ml)
8.1 35.6 14.4
8.3 31.7 18.3
8.5 26.7 23.3
8.7 21.2 28.8
8.9 15.0 35.0

The chemicals used for making the buffer systems were MES [2-(N-morpholino)ethanesulfonic acid; Sigma, M8250, pH 2.7], imidazole (1,3-diazacyclopenta-2,4-diene; BDH, 286874D, pH 9.9), MOPS [3-(N-morpholino)propanesulfonic acid; BDH, 4438321, pH 2.9], HEPES-Na [sodium 4-(2-hydroxy­ethyl)piperazine-1-ethanesulfonate; Melford, B2001, pH 10.4], bicine [N,N-bis(2-hydroxyethyl)glycine; Fluka, 14871, pH 4.9] and Trizma base [proprietary Tris, 2-amino-2-(hydroxy­methyl)-1,3-propanediol; Sigma, T1503, pH 10.6]. The pH was measured at 294 K with an InLab 490 solid-state probe (Mettler–Toledo) to avoid inaccuracies with Tris-containing buffers.

2.5. Stability tests

The stability of the conditions during their development was assessed by checking the turbidity and pH after one week at 293 K, one week at 277 K and another week at 293 K.

2.6. Proteins

For details of the proteins used, please refer to Table 6[link].

Table 6
Details and results of the crystallization trials for 16 samples using MORPHEUS

TEN 200 is a buffer containing 20 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM sodium azide and 200 mM sodium chloride. In the Source column, LMB refers to the MRC Laboratory of Molecular Biology, Cambridge, England, Hutchison to the Hutchison/MRC Research Centre, Cambridge, England, and CPE to the Centre for Protein Engineering, Cambridge, England.

Symbol Protein Concentration (mg ml−1) Molecular weight (kDa) Source Preparation/reference Hits (well numbers) Photo (Fig. 2[link])
TriUB-D Triubiquitin complex 7.0 29.6 LMB, Yogesh Kulathu Manuscript submitted F01, F04, H01, H04 a, b
PI3K-I Pi3-kinase 110delta with inhibitors 4.5 107.0 LMB, Alex Berndt Manuscript submitted C03, C04 c
Scc3 Cohesin subunit 10.0 47.0 LMB, Jan Löwe To be published H07 d
PBD Plk1 polo-box domain 8.7 27.2 Hutchison, Ana J. Narvaez Garcia-Alvarez et al. (2007[Garcia-Alvarez, B., de Carcer, G., Ibanez, S., Bragado-Nilsson, E. & Montoya, G. (2007). Proc. Natl Acad. Sci. USA, 104, 3107-3112.]) B05, D05, D09, E05, F05, F09 e
PBD-P Plk1 polo-box domain with compound 8.7 27.2 Hutchison, Ana J. Narvaez To be published D04 f
DivIVA Tropomyosin 19.2 12.7 LMB, Marian Oliva Manuscript in preparation D07, F07 g
D1-D2 Sm protein complex 16.2 26.9 LMB, Chris Oubridge Kambach et al. (1999[Kambach, C., Walke, S., Young, R., Avis, J. M., de la Fortelle, E., Raker, V. A., Luhrmann, R., Li, J. & Nagai, K. (1999). Cell, 96, 375-387.]) G01 h
ParR Chromosome partitioning 16.0 14.6 LMB, Jeanne Salje Møller-Jensen et al. (2007[Møller-Jensen, J., Ringgaard, S., Mercogliano, C. P., Gerdes, K. & Löwe, J. (2007). EMBO J. 26, 4413-4422.]) G10, G11 i
CRY P53 domain 6.5 27.0 CPE, Joel Kaar & Nicolas Basse Joerger et al. (2006[Joerger, A. C., Ang, H. C. & Fersht, A. R. (2006). Proc. Natl Acad. Sci. USA, 103, 15056-15061.]) D09, E09, G01, G05, G08, G09, G12, H09 j
BAR BAR domain 6.0 29.0 LMB, Helen Kent Peter et al. (2004[Peter, B. J., Kent, H. M., Mills, I. G., Vallis, Y., Butler, P. J., Evans, P. R. & McMahon, H. T. (2004). Science, 303, 495-499.]) A02, C04, C08, C12, G04, G08, G12 k
PAK4G FtsK gamma domain 11.0 7.8 LMB, Jan Löwe Sivanathan et al. (2006[Sivanathan, V., Allen, M. D., de Bekker, C., Baker, R., Arciszewska, L. K., Freund, S. M., Bycroft, M., Löwe, J. & Sherratt, D. J. (2006). Nat. Struct. Mol. Biol. 13, 965-972.]) A01, A05 l
ScVps25 ESCRT II subunit 10.8 23.6 LMB, Olga Perisic Wernimont & Weissenhorn (2004[Wernimont, A. K. & Weissenhorn, W. (2004). BMC Struct. Biol. 4, 10.]) A03, A06, B10, C05, C09, E03, E06, E07, E10, F03, F06, F07, F10 m, n
Ran Ran GTPase 10.0 24.5 LMB, Danguole Ciziene Stewart et al. (1998[Stewart, M., Kent, H. M. & McCoy, A. J. (1998). J. Mol. Biol. 284, 1517-1527.]) G04 o
CNVA Concanavalin A 7.0 26.5 Sigma, L7647 Dissolved in TEN 200 pH 8.5 D02, D06, E02, E06, E10, H02, H06 p
THM Thaumatin 30.0 22.0 Sigma, T7638 Dissolved in deionized water G01, G05, G09 q
LYS Lysozyme 10.0 14.4 Sigma, L6876 Dissolved in deionized water A05, A08, B06, B07, C05, C06, C08, D05, E05, G05, G07, H05 r

2.7. Crystallization trials

MRC crystallization plates (Swissci) containing MORPHEUS (85 µl in the main wells) were prepared on a Mosquito (TTP labtech) or ScreenMaker (Innovadyne) nanolitre liquid handler. Our standard setup for initial screens is to mix equal-volume aliquots of the protein and condition at 297 K, with a 200 nl final volume of drops, and to store the plates at 292 K. Final assessments were made after one week by manual inspection using a high-powered Leica MX-12 stereomicroscope. A drop was considered a crystallization hit when it contained protein crystals larger than 20 µm, so that they could be mounted in a cryoloop for X-ray diffraction.

2.8. Optimization of conditions

Finally, all three components, the ligand mixes, the precipitant mixes and the buffers, are combined using a fixed ratio,

[\eqalign {0.5 \, {\rm stock \,\, precipitants} \, & + \, 0.1 \, {\rm stock \,\,additives} \, \cr & + \, 0.1 \, {\rm buffer \,\,system} \, + \, 0.3 \, {\rm water}.}]

This simple recipe facilitates easy follow-up optimization experiments. As an initial approach, one can simply change the above ratios of the stock solutions. The composition of the buffer systems may be altered during optimization experiments to change the pH. Obviously, all of these optimization experiments are very amenable to automation (Hennessy et al., 2009[Hennessy, D. N., Narayanan, B. & Rosenberg, J. M. (2009). Acta Cryst. D65, 1001-1003.]).

3. Results and discussion

Both well known test proteins and novel samples were tried with MORPHEUS. Table 6[link] shows all the details and results of the crystallization trials performed for 16 samples. Fig. 2[link] shows the different crystal morphologies observed. All the crystals shown represent initial hits, except for Scc3 (domain of sister chromatid cohesion protein 3) and PI3K-I (pi3-kinase p110 in complex with isoform-specific inhibitors) which involved optimization.

[Figure 2]
Figure 2
Light micrographs showing 18 crystals obtained with MORPHEUS (letters refer to Table 6[link], last column). Magnifications differ and crystal sizes vary between 20 and 600 µm.

Importantly, three samples have crystallized exclusively in MORPHEUS and produced no hits from any other screen tried (over 1500 conditions): Scc3, PI3K-I and TriUb-D (triubiquitin in complex with a ubiquitin-binding domain).

The possible specificity of ligand mixes can be spotted easily because of the systematic screen layout: when there are several hits in the same row of MORPHEUS, it means there is specificity to ligands used in the conditions of that row (see samples PI3K-I, ParR, PAK4G and THM). In the same way, specificity to precipitant(s) and pH can easily be noticed (see Fig. 1[link]). For example, most of the hits with the test sample BAR were in conditions that integrate the mix of precipitants developed for MORPHEUS (mix found in columns 4, 8 and 12: 12.5% PEG 1000, 12.5% PEG 3350 and 12.5% MPD).

4. Conclusions

The advantages of designing an initial screen de novo have been demonstrated. MORPHEUS delivers a screen that is easy to make and the conditions are easy to optimize. It contains components that have been selected from crystallized complexes of previously published structures. It also contains a limited number of precipitant mixes that have been selected using local data from the MRC-LMB. MORPHEUS has been successful in crystallizing both known proteins and important new samples.

Ideally, more small molecules with interesting characteristics that are not used in commercially available screens should be investigated, like some polyols (Cohen et al., 1993[Cohen, S., Marcus, Y., Migron, Y., Dikstein, S. & Shafran, S. (1993). J. Chem. Soc. Faraday Trans. 89, 3271-3275.]). An extensive set of amine derivatives, including well known polyamine additives (Ding et al., 1999[Ding, L., Zhang, Y., Deacon, A. M., Ealick, S. E., Ni, Y., Sun, P. & Coleman, W. G. Jr (1999). Acta Cryst. D55, 685-688.]) and aminated amino acids (Matsuoka et al., 2007[Matsuoka, T., Tomita, S., Hamada, H. & Shiraki, K. (2007). J. Biosci. Bioeng. 103, 440-443.]), could form an excellent additive screen with frozen solutions for storage. Also, protein chaperones could be added for some challenging crystallizations (Ostermeier et al., 1995[Ostermeier, C., Iwata, S., Ludwig, B. & Michel, H. (1995). Nat. Struct. Biol. 2, 842-846.]; Tereshko et al., 2008[Tereshko, V., Uysal, S., Koide, A., Margalef, K., Koide, S. & Kossiakoff, A. A. (2008). Protein Sci. 17, 1175-1187.]). In the same spirit, it would be interesting to investigate what could be done with molecules designed to mimic protein–protein interactions (Allen et al., 1998[Allen, J. V., Horwell, D. C., Lainton, J. A. H., O'Neill, J. A. & Ratcliffe, G. S. (1998). Lett. Pept. Sci. 5, 133-137.]).

Acknowledgements

I would like to thank Andrew Turnbull (Cancer Research Technology Ltd.) and Evangelos Papagrigoriou (Insight Research Group) for giving early advice. The development of the screen was sponsored by the MRC-LMB under the supervision of Jan Löwe and Olga Perisic. The frequency of the precipitant mixes was revealed by the LMB screen database (https://www2.mrc-lmb.cam.ac.uk/groups/JYL/WWWrobots/robot.html) developed in collaboration with Paul Hart (MRC-LMB). Data on triubiquitin, pi3-kinase and plk1 complexes were given by Yogesh Kulathu, Alex Berndt and Ana Julia Narvaez, respectively. I would also like to thank everyone at the MRC-LMB for their assistance in trying MORPHEUS and providing test samples for two years before its commercialization. Conflicting commercial interest: I hereby state that I have a conflicting commercial interest, in that the MORPHEUS crystallization screen has been commercialized by Molecular Dimensions Ltd (https://www.moleculardimensions.com) under an exclusive licence to MRC Technology.

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