Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future

Abstract

The crystallisation of proteins and macromolecular complexes is an essential step for studying the three‐dimensional structure of the molecular components that make up living cells. Protein crystals are formed by the self‐assembly of protein molecules into an ordered, periodic lattice arrangement. Crystallisation is initiated by reducing the solubility of the protein sample by the addition of precipitating agents such as salts and polyethylene glycols (PEG). This creates a supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, the protein‐rich phase can adopt the form of an ordered protein crystal. The most common technique used for crystallising proteins is vapour diffusion, which relies on the loss of water from the growth solution during equilibration with a reservoir of precipitant. Protein crystals can be extremely difficult to grow and often require exhaustive screening of many different parameters such as concentration and type of precipitant, pH, temperature, additives and variations in protein construct.

Key Concepts:

  • A detailed knowledge of the molecular structure of proteins and macromolecular complexes is essential for understanding how the molecular machinery of living cells operates.

  • Crystallography is the most prevalent method for studying the structures of proteins and macromolecular complexes at the heart of the cellular machinery.

  • Protein crystals are formed by the self‐assembly of large polypeptide chains into an ordered, regular and repeating lattice arrangement of protein molecules.

  • Crystallisation of proteins is generally carried out by the addition of precipitating agents to the protein solution, which results in the generation of a metastable, supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, this protein‐rich phase can adopt the form an ordered protein crystal.

  • Several experimental techniques are available to crystallise proteins, including vapour diffusion, microbatch crystallisation under oil, microdialysis, free‐interface diffusion and crystallisation in Lipidic Cubic Phase (LCP).

  • The most commonly used crystallisation technique is vapour diffusion, which relies on the diffusion of water from the crystal growth solution to a precipitant reservoir.

  • Even the most beautiful protein crystals do not necessarily diffract X‐rays. Many protein crystals suffer from disordered regions as a result of thermal or static displacement of atoms. Disorder is often a result of flexible domains in the protein that may adversely affect how the protein molecules are packaged together in the crystal.

  • Recombinant DNA techniques can be used to engineer protein constructs that are more amenable to crystallisation by increasing the stability and/or solubility of the protein. Additionally, the amino acid residues on the surface of the protein can be modified to alter the intermolecular surface contacts within the crystal.

  • Complexes of proteins bound to small molecule substrates such as cofactors or potential drugs can be crystallised. Additionally, very large complexes between multiple proteins or with other macromolecules such as RNA or DNA can be crystallised.

Keywords: protein crystals; protein crystallography; protein crystallisation; X‐ray diffraction; structural biology; protein structure; macromolecular complexes

Figure 1.

The nature of protein crystals. (a) Protein crystals are formed by self‐organisation of protein molecules into an ordered, three‐dimensional network. In this example, the proteins are held together in a regular repeating fashion by a series of weak intermolecular interactions shown by the red, green and blue arrows. (b) Crystal of protein PC50891A grown in a nanodroplet that was setup by an automated robotics system. (c) Lysozyme crystal produced by hand pipetting of much larger volumes. Images of crystals acquired using a digital camera attached to an optical microscope at a magnification of approximately 80x. Adapted from Rupp ().

Figure 2.

Protein solubility diagram and nucleation process. The three main regions observed during the course of protein crystallisation are highlighted; stable protein solution (blue), metastable, supersaturated region (green) and unstable region (red). The spontaneous formation of crystallisation nuclei potentially leading to protein crystals occurs in the metastable region at higher supersaturation. Adapted from Rupp ().

Figure 3.

Common protein crystallisation methods. The most commonly employed methods include (a) hanging drop vapour diffusion and (b) sitting drop vapour diffusion. Hanging drop methods are typically employed for low‐throughput manual setups, whereas sitting drop methods are more amenable for robotics and automated setups. (c) Microbatch, (d) microdialysis and (e) free‐interface diffusion are less commonly used, but are powerful tools in the crystallographer's arsenal for proteins recalcitrant to crystallisation using standard techniques. (f) Microbatch Lipidic Cubic Phase (LCP) experiment used for crystallising membrane proteins. The LCP bolus is shown in red sandwiched between a glass cover slide and the crystallisation plate. (g) Workflow of a typical hanging drop vapour diffusion experiment. Adapted from Rupp ().

Figure 4.

Path of a successful vapour diffusion experiment through the protein solubility diagram. Example illustrates the principles of a typical hanging drop vapour diffusion experiment. Equal sized drops of protein (p; dark green circle) and crystallisation cocktail (c; blue circle) are pipetted onto a cover slide, inverted and sealed over a larger reservoir of crystallisation cocktail. This results in a starting drop (S, light‐green circle) with protein and crystallisation cocktail concentrations half that of the original (p/2 and c/2, respectively). As a result, water vapour diffuses out of the starting drop in an attempt to reestablish equilibrium. This gradually increases both the protein and precipitant concentrations in the drop, and once the solution is sufficiently supersaturated, nucleation of a crystal may occur (1) and if conditions are suitable, crystal growth may continue (2 through 4). Adapted from Rupp ().

Figure 5.

Increasing size and resolution range of macromolecular complexes in the PDB. Panel (a), (b) and (c) show the distribution of resolution, size and number of polymers for each year of PDB depositions, respectively. Some of the early PDB entries are shown in panel (d) along with some other structures of note from Table . Adapted from Jones ().

close

References

Abad‐Zapatero C (2012) Notes of a protein crystallographer: on the high‐resolution structure of the pdb growth rate. Acta Crystallographica Section D 68: 613–617.

Andersson KM and Hovmoller S (2000) The protein content in crystals and packing coefficients in different space groups. Acta Crystallographica Section D, Biological crystallography 56: 789–790.

Beck F, Unverdorben P, Bohn S et al. (2012) Near‐atomic resolution structural model of the yeast 26s proteasome. Proceedings of the National Academy of Sciences of the USA 109: 14870–14875.

Berman HM (2008) The protein data bank: a historical perspective. Acta Crystallographica. Section A, Foundations of crystallography 64: 88–95.

Bernstein FC, Koetzle TF, Williams GJ et al. (1977) The protein data bank: a computer‐based archival file for macromolecular structures. Journal of Molecular Biology 112: 535–542.

Bhattacharya A (2009) Protein structures: structures of desire. Nature 459: 24–27.

Carter CW Jr and Carter CW (1979) Protein crystallization using incomplete factorial experiments. Journal of Biological Chemistry 254: 12219–12223.

Chapman HN, Fromme P, Barty A et al. (2011) Femtosecond X‐ray protein nanocrystallography. Nature 470: 73–77.

Chayen NE (2009) High‐throughput protein crystallization. Advances in Protein Chemistry and Structural Biology 77: 1–22.

Cudney R, Patel S, Weisgraber K et al. (1994) Screening and optimization strategies for macromolecular crystal growth. Acta Crystallographica Section D, Biological Crystallography 50: 414–423.

Féthière J (2007) Three‐dimensional crystallization of membrane proteins. In: Walker J and Doublié S (eds) Macromolecular Crystallography Protocols, pp. 191–223. New York: Humana Press.

Gilman JJ (2009). Chemistry and physics of mechanical hardness. New York: Wiley.

Goldschmidt L, Eisenberg D and Derewenda ZS (2014) Salvage or recovery of failed targets by mutagenesis to reduce surface entropy. Methods in Molecular Biology (Clifton, NJ) 1140: 201–209.

Gorrec F (2009) The morpheus protein crystallization screen. Journal of Applied Crystallography 42: 1035–1042.

Hoelz A, Debler EW and Blobel G (2011) The structure of the nuclear pore complex. Annual Review of Biochemistry 80: 613–643.

Jancarik J and Kim S‐H (1991) Sparse matrix sampling: a screening method for crystallization of proteins. Journal of Applied Crystallography 24: 409–411.

Jones N (2014) Crystallography: atomic secrets. Nature 505: 602–603.

Julien JP, Cupo A, Sok D et al. (2013) Crystal structure of a soluble cleaved hiv‐1 envelope trimer. Science (New York, NY) 342: 1477–1483.

Kendrew JC, Bodo G, Dintzis HM et al. (1958) A three‐dimensional model of the myoglobin molecule obtained by X‐ray analysis. Nature 181: 662–666.

Landau EM and Rosenbusch JP (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proceedings of the National Academy of Sciences of the USA 93: 14532–14535.

Luft JR, Wolfley JR, Said MI et al. (2007) Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature. Protein science: A Publication of the Protein Society 16: 715–722.

Marchler‐Bauer A, Zheng C, Chitsaz F et al. (2013) Cdd: conserved domains and protein three‐dimensional structure. Nucleic Acids Research 41: D348–D352.

McPherson A (1982) The Preparation and Analysis of Protein Crystals. New York, NY: Wiley.

McPherson A (1998) Crystallization of Biological Macromolecules. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

McPherson A and Cudney B (2006) Searching for silver bullets: an alternative strategy for crystallizing macromolecules. Journal of Structural Biology 156: 387–406.

Newman J, Egan D, Walter TS et al. (2005) Towards rationalization of crystallization screening for small‐ to medium‐sized academic laboratories: the pact/jcsg+ strategy. Acta Crystallographica Section D 61: 1426–1431.

Peat TS, Christopher JA and Newman J (2005) Tapping the protein data bank for crystallization information. Acta Crystallographica Section D 61: 1662–1669.

Protein Data Bank (1971) Protein data bank. Nature New Biology 233: 223.

Pryor EE Jr, Wozniak DJ and Hollis T (2012) Crystallization of Pseudomonas aeruginosa amrz protein: development of a comprehensive method for obtaining and optimization of protein–DNA crystals. Acta Crystallographica Section F, Structural Biology and Crystallization Communications 68: 985–993.

Radaev S and Sun PD (2002) Crystallization of protein–protein complexes. Journal of Applied Crystallography 35: 674–676.

Rasmussen SG, Choi HJ, Rosenbaum DM et al. (2007) Crystal structure of the human beta2 adrenergic G‐protein‐coupled receptor. Nature 450: 383–387.

Rupp B (2009) Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology, 1st edn. New York, NY: Garland Science.

Santarsiero BD, Yegian DT, Lee CC et al. (2002) An approach to rapid protein crystallization using nanodroplets. Journal of Applied Crystallography 35: 278–281.

Slabinski L, Jaroszewski L, Rychlewski L et al. (2007) Xtalpred: a web server for prediction of protein crystallizability. Bioinformatics 23: 3403–3405.

Spraggon G, Pantazatos D, Klock HE et al. (2004) On the use of dxms to produce more crystallizable proteins: structures of the t. Maritima proteins tm0160 and tm1171. Protein Science: A Publication of the Protein Society 13: 3187–3199.

Terwilliger TC, Stuart D and Yokoyama S (2009) Lessons from structural genomics. Annual Review of Biophysics 38: 371–383.

Tickle I, Sharff A, Vinkovic M et al. (2004) High‐throughput protein crystallography and drug discovery. Chemical Society Reviews 33: 558–565.

Voorhees RM, Weixlbaumer A, Loakes D et al. (2009) Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70s ribosome. Nature Structural and Molecular Biology 16: 528–533.

Walter TS, Diprose JM, Mayo CJ et al. (2005) A procedure for setting up high‐throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallographica Section D, Biological crystallography 61: 651–657.

Wernimont A and Edwards A (2009) In situ proteolysis to generate crystals for structure determination: an update. PLoS One 4: e5094.

Further Reading

Bergfors T (2009) Protein Crystallization. San Diego, CA: International University Line.

Chayen N (2007) Protein Crystallization Strategies for Structural Genomics. San Diego, CA: International University Line.

Doublie S (2007) Macromolecular Crystallography Protocols. Preparation and Crystallization of Macromolecules, vol. 1. Totowa, NJ: Humana Press.

Ducruix A and Giege R (1999) Crystallization of Nucleic Acids and Proteins. Oxford: Oxford University Press.

McPherson A (1989) Macromolecular crystals. Scientific American 260: 62–69.

McPherson A (1990) Current approaches to macromolecular crystallization. European Journal of Biochemistry 189: 1–23.

McPherson A (2004) Introduction to protein crystallization. Methods 34: 254–265.

Rupp B (2009) Biomolecular crystallography: principles. Practice, and Application to Structural Biology, 1st edn. New York, NY: Garland Science.

Sauter C, Lorber B, McPherson A and Giege R (2012) Crystallization. General methods. In: Arnold E, Himmel DM and Rossmann MG (eds) International Tables for Crystallography. Volume F: Crystallography of Biological Macromolecules, 2nd edn, pp. 99–121. Chichester, NH: Wiley.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Deller, Marc C, and Rupp, Bernhard(Aug 2014) Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002718.pub2]