On the Quality of Protein Crystals Grown under Diffusion Mass-transport Controlled Regime (I)

Abstract

It has been previously shown that the diffraction quality of protein crystals strongly depends on mass transport during their growth. In fact, several studies support the idea that the higher the contribution of the diffusion during mass transport, the better the diffraction quality of the crystals. In this work, we have compared the crystal quality of two model (thaumatin and insulin) and two target (HBII and HBII-III) proteins grown by two different methods to reduce/eliminate convective mass transport: crystal growth in agarose gels and crystal growth in solution under microgravity. In both cases, we used identical counterdiffusion crystallization setups and the same data collection protocols. Additionally, critical parameters such as reactor geometry, stock batches of proteins and other chemicals, temperature, and duration of the experiments were carefully monitored. The diffraction datasets have been analyzed using a principal component analysis (PCA) to determine possible trends in quality indicators. The relevant indicators show that, for the purpose of structural crystallography, there are no obvious differences between crystals grown under reduced convective flow in space and convection-free conditions in agarose gel, indicating that the key factor contributing to crystal quality is the reduced convection environment and not how this reduced convection is achieved. This means that the possible detrimental effect on crystal quality due to the incorporation of gel fibers into the protein crystals is insignificant compared to the positive impact of an optimal convection-free environment provided by gels. Moreover, our results confirm that the counterdiffusion technique optimizes protein crystal quality and validates both environments in order to deliver high quality protein crystals, although other considerations, such as protein/gel interactions, must be considered when defining the optimal crystallization setup.

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin

“…Crystals obtained by vapor diffusion were protected with a variety of cryo-protection strategies, from naked crystal diffraction to several standard cryo-protectants tested at different concentrations and soaking times. To provide better control of nucleation and crystal growth, we turned to several non-conventional crystallization techniques [17][18][19][20], namely counter-diffusion in agarose gels in several of its possible setups, which also allows the exploration of the supersaturation rate. These efforts have yielded good-looking crystals, but their stability is compromised once they are removed from their growth medium, making it impossible to proceed to X-ray diffraction.…”

Multivariate Analysis Applications in X-ray Diffraction

“…Crystal recognition is a key step in high-throughput Protein Crystallography initiatives, where high-throughput crystallization screening is implemented, which demand for the systematic analysis of crystallization results. 42 diffraction datasets related to protein crystals grown under diffusion mass-transport controlled regime have been analyzed by PCA to determine possible trends in quality indicators [85]. In reference [86] neural networks were used to assess crystal quality directly from diffraction images, i.e., without using quality indicators.…”

Production of Cross-Linked Lipase Crystals at a Preparative Scale

“…The use of gels to mimic microgravity conditions (convection-free environment, no sedimentation, etc.) and to improve the diffraction quality of biomacromolecules crystals 48 − 50 ends up proving that protein crystals incorporated the gel matrix during their growth, producing new composite materials of improved properties named reinforced protein crystals. 51 − 55 The nature of the gel has also been used to modify the properties of CLECs.…”

“…The use of gels to mimic microgravity conditions (convection-free environment, no sedimentation, etc.) and to improve the diffraction quality of biomacromolecules crystals − ends up proving that protein crystals incorporated the gel matrix during their growth, producing new composite materials of improved properties named reinforced protein crystals. − The nature of the gel has also been used to modify the properties of CLECs. For example, lysozyme crystals grown in Fmoc-dipeptide hydrogels loaded with single-walled carbon nanotubes were used to produce reinforced cross-linked lysozyme crystals able to conduct electricity .…”