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

José A Gavira,Emilio Melero,Juan Manuel García-Ruíz,Fermín Otálora,Alexander E.S Van Driessche,Luis A González-Ramírez

doi:10.3390/cryst10020068

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.

Highlights

Obtaining crystals of sufficient quality is essential for obtaining good diffraction data and building accurate 3D structural models of both small and macromolecules
We have conclusively shown that counterdiffusion methods produce crystals of higher quality than conventional convective crystals [27], but after more than 25 years of investigation, there is still no clear picture of the possible advantages of microgravity conditions over on-ground gel growth in terms of protein crystal quality [4,7,46,47]
This suggests that this dimension could be relevant in terms of crystal quality grown in gels

Summary

Introduction

Obtaining crystals of sufficient quality is essential for obtaining good diffraction data and building accurate 3D structural models of both small and macromolecules. The formation of a stable depletion zone in diffusive setups minimizes and stabilizes the supersaturation at the crystal/solution interface, reducing and making constant the growth rate, which kinetically favors the arrangement of molecules at the crystal surface and allows for efficient transport of impurities out of it. This is generally accepted as a factor enabling crystal perfection [17,18,19,20]. It was concluded that the diffusive mass-transport conditions created during microgravity experiments are a key factor in obtaining good quality protein crystals [21,22]

Methods

Results

Conclusion