Log in

Login to your account

Username
Password *

Thermofluor & Crystallization likelihood

 

 

 

The identification of crystallization conditions for biological molecules largely relies on a trial-and-error process in which a number of parameters are explored in large screening experiments. Currently, construct design and sample formulation are recognized as critical variables in this process and often a number of protein variants are assayed for crystallization either sequentially or in parallel, which adds complexity to the screening process. Significant effort is dedicated to sample characterization and quality-control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. However, large-scale studies relating crystallization success to sample properties are generally lacking.

 

In this study(1), the thermal stability of 657 samples was estimated using a simplified Thermofluor assay. These samples were also subjected to automated vapour-diffusion crystallization screening under a constant protocol.

 

Analysis of the data shows that:

  • samples with an apparent melting temperature (Tm) of 45°C or higher crystallized in 49% of cases, 
  • the crystallization success rate decreased rapidly for samples with lower Tm. Only 23% of samples with a Tm below 45°C produced crystals.

 

Based on this work, when the estimated Tm is lower than the critical value of 45°C, we recommend to undertake the following actions:

  • If the selected incubation temperature was 20°C, we advise to perform a new screening at  4°C to maximize crystallization likelihood (1).
  • If the selected incubation temperature was 4°C and no crystal was obtained, we advise you to explore the possibility to screen for buffer conditions that could increase the thermal stability of your sample (2) See for example our protocols available for 96 wells stability optimization screens
  • If none of this works, consider assaying new constructs.

 

 

Hence, a simple method for estimation of the crystallization likelihood of biological samples is proposed. This method is easy, rapid and consumes very small amounts of sample. The results of this assay can be used to determine optimal incubation temperatures for crystallization experiments or to prioritize certain constructs. More generally, this work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects. 

 

 

The HTX Lab performs Thermofluor assays systematically on every sample processed at the crystallization platform (3). The Thermofluor graph is accessible through CRIMS (Under "User" menu "My samples").

 

 

tsa image

Fig 1: Thermofluor curve in CRIMS

 

References

1. Dupeux et al. Acta Cryst. (2011), D67; 915–919. PMID:22101817

2. Ericson et al.  Anal. Biochem. (2006), 357; 289-298. PMID: 16962548

3. Mariaule et al. Methods Mol Biol. (2014), 1091:189-95. PMID:24203333