Tips and Tricks in Crystallography

Pre-crystallization Modification

Jerry Alexandratos: Effect of Dry Ice on Protein Stability. When materials are 
stored on dry ice, no matter what container is used or how well sealed, CO2 can
infiltrate into the head spece over the frozen sample. Upon traveling, the CO2 will
dissolve into the solution and lower pH, which can affect many proteins assays or
protein stability or solubility (especially near the protein's isoelectric point). The
simplest miethods for eliminating this problem are to vent protein containers before
thawing. or storing the samples in a -70°C freezer for at least four days before
thawing (for a sysmatic analysis by Murphy et al., see PMID 23538862).
Xinhua Ji (NCI):Lysine Methylation
It appears that lysine methylation should be considered a routine step not only in traditional (low-throughput) crystallization trials but also for structural genomics (high-throughput) attempts. Walter and co-workers outlined a detailed protocol with ten examples in their recent article (Structure 14:1617-1622, 2006). The protocol was derived from previously published method by Rayment and co-workers (Science 261:50-58, 1993) and Rayment (Methods Enzymol. 276:171-179, 1997).  A recent success within the NIH X-ray Diffraction Group was reported by Schubot and Waugh (Acta Cryst. D60:1981-1986, 2004), showing that the method was pivotal in the de novo crystallization of a ternary complex that contains three protein molecules!

Dr. David Waugh (NCI): When an otherwise well-behaved protein fails to crystallize, what do you (suggest to) do? It is not uncommon for proteins to have disordered termini, which may impede the formation of crystals. Therefore, when an otherwise well-behaved protein fails to crystallize, the first thing we do is subject it to limited proteolysis with thermolysin. We prefer thermolysin because its major specificity determinant is a hydrophobic residue in the P1’ position. Hydrophobic residues occur much less frequently than arginine and lysine in solvent-exposed loops of proteins. Consequently, thermolysin is less likely than trypsin or chymotrypsin to yield misleading results. We have also found that in general, digestion patterns generated by thermolysin are cleaner, which simplifies the identification of the digestion products. Metastable digestion products can be identified by mass spectrometry and N-terminal amino acid sequencing, and then new vectors can be constructed to overproduce the truncated polypeptides. Secondary structure prediction and sequence alignments can sometimes used to make educated guesses about the locations of domain boundaries when the proteolysis approach fails to yield definitive results. We also recommend trying reductive alkylation with formaldehyde and dimethylamine-borane complex (Rayment, I. Methods Enzymol. 276, 171-179 [1997]). The net result of this reaction is the dimethylation of all accessible lysine side chains (and the N-terminal amino group). Although this does not change the intrinsic charge of a protein, it may alter its isoelectric point slightly. One rational behind this strategy is that dimethylation of lysine side chains will reduce their interaction with solvent, thereby causing them to adopt more “ordered” conformations that may facilitate crystallization. Reductive methylation also frequently reduces the solubility of proteins, and so it may be a good approach to try when mostly clear drops are obtained from crystallization screens even with very concentrated solutions of protein. The nice thing about this approach is that it can be performed on the existing sample of protein (i.e., no new constructs need to be made). Surface entropy reduction mutagenesis, a strategy pioneered by Zygmunt Derewenda and coworkers, is another option. In this method, linear clusters of amino acid side chains with high conformational entropy (e.g., Lys and Glu), which are presumed to lie on the surface of the protein, are replaced by methyl groups (Ala) in an effort to create new epitopes that will facilitate crystallization. A growing number of proteins have been crystallized in this manner, suggesting that the method may be of general utility. Yet, because is impossible to predict which cluster mutant(s) will crystallize, the probability of a successful outcome is proportional to the number of mutants that are screened. Consequently, surface entropy reduction mutagenesis can be a very labor intensive undertaking. Finally, if the opportunity exists, working with multiple orthologs of a target protein generally improves the odds of obtaining crystals.

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