3.1. Diffusive mixing kinetics increased crystallization productivity relative to vapour diffusion for well characterized proteins
Thaumatin, catalase and myoglobin were screened with a subset of solutions from the sparse-matrix screens Crystal Screen (48 conditions) and JCSG-plus (48 conditions) in parallel in sitting-drop vapour-diffusion plates and Crystal Former devices. All three proteins yielded crystals in both methods. Remarkably, there were significant differences in the identities and numbers of crystallization conditions for the Crystal Former and sitting-drop experiments. For trials with the Crystal Former, crystals were obtained for 5, 28 and 8% of all conditions for thaumatin, catalase and myoglobin, respectively. The success rates were 1, 7 and 1% for thaumatin, catalase and myoglobin, respectively, using sitting-drop vapour diffusion. Consequently, a fourfold to eightfold increase in success for initial crystallization trials was observed in the Crystal Former (Table 1).
Relative increase in crystallization outcomes for catalase, myoglobin and thaumatin using the Crystal Former and vapour-diffusion methods
A comparison of the identified crystallization conditions revealed striking differences between vapour diffusion and the microfluidic device (Fig. 2). Approximately 40% of the conditions identified by vapour diffusion were unique to that method and were not captured in the Crystal Former trials. Similarly, 90% of the crystals grown in the Crystal Former were not identified by the sitting-drop experiments, highlighting the advantages of the Crystal Former in sampling the protein phase space.
Figure 2 Each protein was screened by Crystal Screen (Hampton Research) and JCSG-plus 1 (Molecular Dimensions, UK) crystal screening using both sitting-drop vapour diffusion and the Crystal Former. Crystallization conditions identified with the Crystal Former (more ...)
To verify that the crystals obtained using the Crystal Former were indeed protein crystals, a representative subset was harvested and their respective diffraction was analyzed on the X6A beamline at the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, New York. USA). For each mounted crystal, several frames were collected in order to verify that these were indeed protein crystals. The maximal resolution obtained from these crystals ranged from 2.8 to 1.1 Å, highlighting the good diffraction quality of the extracted crystals.
3.2. Harvesting and structure determination of thaumatin
Thaumatin crystals were grown and prepared for data collection as described previously (Fig. 3
). A single crystal was mounted in a cryoloop and cooled in liquid nitrogen. A complete set of diffraction data was collected and the structure was determined by molecular replacement (Fig. 3
). Electron density was apparent for 206 of the 207 amino-acid residues. The thaumatin structure was refined to 1.25 Å resolution, with R
values of 15.4% and 16.9%, respectively (Table 2). A single thaumatin monomer was modelled in the asymmetric unit, along with 201 water molecules and ten tartrate ions. This crystal form was isomorphous to 11 structures previously deposited in the Protein Data Bank (1lr3
). For these depositions, the resolution ranged from 1.05 to 2.3 Å and the R
values spanned the range 15.2–25%. As observed in the thaumatin structure reported here, no density was observed for the C-terminal alanine in PDB entries 2blr
. The remaining entries report unambiguous density for all 207 residues of thaumatin.
Figure 3 (a) Crystals of thaumatin were grown from 0.8 M potassium sodium tartrate, 0.1 M HEPES pH 7.5 in the Crystal Former. Here they are shown under polarized light. (b) Representative 2F
o − F
c electron (more ...)
Data collection and structure refinement
In situ data collection and structure determination for lysozyme
The Crystal Formers were also assessed for their compatibility with in situ X-ray analysis on the X6A beamline (National Synchrotron Light Source, Brookhaven National Laboratory). The Crystal Former was mounted lengthwise with the microchannels perpendicular to the goniometric head. Not only could the Crystal Formers be mounted for the identification of protein crystals, but a complete data set for lysozyme crystals contained within the microchannels could be collected in situ at room temperature (Fig. 3
c, Table 2). The crystal structure of lysozyme was determined at 1.65 Å resolution with R and R
free values of 17.2% and 22.2% for the final refined structure. These crystals were isomorphous to 78 previous PDB entries. The resolution of previously deposited lysozyme structures ranged from 0.94 to 3.9 Å, with R
free values ranging from 14.5 to 32.3%.
3.4. Compatibility of the Crystal Former with in situ UV analysis
A commonly used approach for imaging protein-crystallization experiments is UV fluorescence. The amino acid tryptophan emits light at approximately 360 nm when excited with light of 280 nm; hence, if a target protein contains the amino acid tryptophan it should be possible to distinguish target protein crystals from precipitant crystals based on fluorescence. Lysozyme crystals (10–100 µm) were grown in the Crystal Former and the channels were imaged by placing the microfluidic device in a slide holder mounted on the XY stage of a UVEX microscope (JAN Scientific, USA). Fluorescence from the larger crystals (>100 µm) could be visualized with the 5× objective, whereas the small crystals (~10 µm) could only be seen with the 15× objective by virtue of the higher fluorescence excitation, collection efficiency and higher spatial resolution of the higher power objective (Fig. 3
d). The background signal and UV absorption from the material of the Crystal Former was sufficiently low that fluorescence from even the smallest crystals could be detected reliably.