Setting aside possible advantages in phasing and structure determination that might arise with centrosymmetric protein crystals, there is now good reason to believe that racemic crystallography could have a much more profound impact, by dramatically improving the ease with which macromolecular crystals can be obtained; the crystallization problem remains the most serious and most vexing obstacle in macromolecular crystallography. The prediction in 1995 that crystallization from a racemic mixture might provide a critical advantage arose largely as a surprise while working on a mathematical explanation for why some special crystal space groups are preferred so strongly over others by proteins when they crystallize (i.e. in ordinary experiments involving only the natural biological hand). The preferences for different crystal space groups vary by more than two orders of magnitude, so understanding that phenomenon has potentially important implications for the crystallization problem as a whole. The paper by Stephanie Wukovitz in 1995 offered a mathematical explanation for the space group preference phenomenon; i.e. it explained why P212121 is by far the most commonly observed crystal space group for (ordinary chiral) proteins. Reaching farther, it was also noted at that time that the theory predicted that even more dominant crystal space groups existed among those that are only possible when using a racemic mixture. Two predictions were made: that proteins would crystallize with greater ease from racemic mixtures owing to the existence of especially favored racemic crystal symmetries, and that P1(bar) would be the dominant space group observed.

The invention of native chemical ligation methods by Phil Dawson and Stephen Kent in the early 1990's opened up prospects for chemically synthesizing larger protein molecules. Kent and co-workers have since tested racemic crystallography on a wide range of protein molecules. Current data provide strong support for the idea that proteins do crystalize with realtive ease from synthetic racemic mixtures (and most often in P1(bar) as predicted).

References:

• Yeates TO, Kent SB. (2012). Racemic protein crystallography. Annu Rev Biophys. 2012. 41:41-61. [Abstract]
  Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.
• Sawaya MR, Pentelute BL, Kent SB, Yeates TO. (2012). Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data. Acta Crystallogr. D Biol. Crystallogr.. Jan 2012. 68(Pt 1):62-8. [Abstract]
  Racemic protein crystallography offers two key features: an increased probability of crystallization and the potential advantage of phasing centric diffraction data. In this study, a phasing strategy is developed for the scenario in which a crystal is grown from a mixture in which anomalous scattering atoms have been incorporated into only one enantiomeric form of the protein molecule in an otherwise racemic mixture. The structure of a protein crystallized in such a quasi-racemic form has been determined in previous work [Pentelute et al. (2008), J. Am. Chem. Soc. 130, 9695-9701] using the multiwavelength anomalous dispersion (MAD) method. Here, it is shown that although the phases from such a crystal are not strictly centric, their approximate centricity provides a powerful way to break the phase ambiguity that ordinarily arises when using the single-wavelength anomalous dispersion (SAD) method. It is shown that good phases and electron-density maps can be obtained from a quasi-racemic protein crystal based on single-wavelength data. A prerequisite problem of how to establish the origin of the anomalous scattering substructure relative to the center of pseudo-inversion is also addressed.
• Banigan JR, Mandal K, Sawaya MR, Thammavongsa V, Hendrickx AP, Schneewind O, Yeates TO, Kent SB. (2010). Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography. Protein Sci.. Oct 2010. 19(10):1840-9. [Abstract]
  The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.
• Pentelute BL, Mandal K, Gates ZP, Sawaya MR, Yeates TO, Kent SB. (2010). Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography. Chem. Commun. (Camb.). Nov 2010. 46(43):8174-6. [Abstract]
  Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.


• Wukovitz SW, Yeates TO. (1995). Why protein crystals favour some space-groups over others. Nat. Struct. Biol.. Dec 1995. 2(12):1062-7. [Abstract]
  One of the most puzzling observations in protein crystallography is that the various space-group symmetries occur with striking non-uniformity. Molecular close-packing has been invoked to explain similar observations for crystals of small organic compounds, but does not appear to be the dominant factor for proteins. Instead, we find that the observed frequencies for both two- and three-dimensional crystals can be explained by an entropic model. Under a requirement for connectivity, the favoured space groups are simply less restrictive than others in that they allow the molecules more rigid-body degrees of freedom and can therefore be realized in a greater number of ways. This result underscores the importance of the nucleation event in crystallization and leads to specific ideas for crystallizing water-soluble and membrane proteins.