A thermal stability assay can help to estimate the crystallization likelihood of biological samples.

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, 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 (T(m)) of 318 K or higher crystallized in 49% of cases, while the crystallization success rate decreased rapidly for samples with lower T(m). Only 23% of samples with a T(m) below 316 K produced crystals. Based on this analysis, 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.

Automated analysis of vapor diffusion crystallization drops with an X-ray beam.

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.

Crystallization and preliminary crystallographic analysis of the motor domain of human kinetochore-associated protein CENP-E using an automated crystallization procedure.

Human centromere-associated protein E, a member of the kinesin superfamily, is a microtubule-dependent motor protein involved in cell division that has been localized transiently to the kinetochore. The protein is thought to be responsible for the correct attachment and positioning of chromosomes to the mitotic spindle during the metaphase. The 312 kDa protein comprises four different domains. In this study, the focus was on the N-terminal motor domain, which includes the ATP-binding site and a region for microtubule binding. Crystals of the CENP-E motor domain have been obtained by high-throughput crystallization screening using an automated TECAN crystallization robot. The crystals (737 x 132 x 79 microm) belong to the space group P2(1), with unit-cell parameters a = 49.35, b = 83.70, c = 94.16 angstroms, beta = 103.05 degrees. They diffract to 2.1 angstroms resolution using synchrotron radiation.