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The effect of osmotic shock on tetragonal lysozyme crystals is experimentally demonstrated. The network of channels and the charge of the crystal are responsible for a Gibbs-Donnan effect. This plays a critical role in the exchange of water, which leads to the formation of cracks.
research papers
The crystallization, cryoprotection and heavy-atom derivatization of insulin have been optimized using the counter-diffusion technique in a restricted geometry. The protein crystals obtained were immediately used for in situ X-ray diffraction, phase determination by SAS and electron-density map calculation without crystal manipulation.
research papers
This paper reports experimental results and modelling on the crystallisation processes induced by counter diffusion method of a precipitant agent in a lysozyme protein solution. Comparison between experimental observations and numerical simulations in the presence of convection and sedimentation and without them (suppressed using gel) provides a validation of the model. Different values of the initial protein concentration are used, in order to investigate the effects of supersaturation conditions on the process, and in particular on nucleation. The model and the experimental approach may represent a useful methodology for the determination of the parameters and conditions that may lead to protein crystallisation. A Mach-Zehnder interferometer is used to monitor the transport dynamics in situ in the fluid phase by observing the compositional field. The effect of the solute transport gives rise to a "nucleation front" that propagates inside the protein solution. The crystal formation, caused by progressing of the front, results in a modulation in time and in space (similar to Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. Both experimental observation and numerical modelling show spatial and size distributions of crystals that demonstrate comparable evidences of the phenomena.
research papers
Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.
research papers
The crystallisation pressure exerted by protein crystals growing in agarose gel does not disrupt the gel network. However, protein crystals trap agarose fibres when they grow in agarose gels. The fibres of agarose are distributed randomly in the crystals explaining why they do not appreciably affect the diffraction quality of the crystal
research papers
Mach-Zehnder interferometry is applied to quantitatively characterize growth of lysozyme crystals in microgravity. Experiments were performed by the Free Interface Diffusion technique into APCF FID reactors using large seeds. Tracking of the experiments using interferometry allowed to monitor the onset of supersaturation and the seed growth. A large and stable concentration depletion zone around the growing crystal developed, whose time evolution was analyzed. The interferograms were analyzed taking into account finite thickness of the cell by integrating the concentration over the straight lines through the optical path. It was concluded that there may be a quasi-steady state growth mode at the stage when the spacial concentration distribution did not change but its absolute value over all the cell was slowly diminishing. From this portion of the data, an estimate was made of the dimensionless parameter βR/D where β is the face kinetic coefficient, R is the effective crystal size and D is the lysozyme diffusivity in solution, as followed from the steady state model. For the assumed quasi steady state data portion, the parameter varies between 0.7 and 0.9 suggesting mixed diffusion-interface kinetic controlled growth.
