Abstract
A large high-quality crystal is required to specify the positions of H atoms in neutron structural analysis. Consequently, several methods have been proposed for obtaining such large crystals, and theoretical considerations for growing them have been presented. However, further investigation is required to obtain a numerical model that can provide quantitative experimental conditions for obtaining a single large crystal. In the case of protein crystallization experiments, the amount of sample is often limited. Therefore, it is more realistic to make a rough estimation from a small number of experiments. This paper proposes a method of estimating the optimum experimental conditions for the growth of large protein crystals by performing a small number of experiments using a micro-batch method and reporting a numerical model based on nucleation theory and a linear approximation of the crystal-growth rate. Specifically, micro-batch experiments are performed to provide the empirical parameters for the model and to help to estimate the conditions for the growth of a crystal of a predetermined size using a certain sample concentration and volume. This method is offered as a step on the path towards efficiently and rationally producing large crystals that can be subjected to neutron diffraction without depending on luck or on performing many experiments. It is expected to contribute to drug design and the elucidation of protein molecular functions and mechanisms by obtaining positional information on H atoms in the protein molecule, which is an advantage of neutron diffraction.
Highlights
Neutron protein crystallography, a powerful neutron diffraction technique for investigating protein chemistry, has elicited considerable interest among academics and pharmaceutical companies
We developed a method for estimating the optimum experimental conditions for the growth of a large crystal using a certain volume of sample solution
When the sodium chloride concentration was 0.4 M, which is the same condition as the experimental condition in Table 1, no crystals grew, even after six months, in 5 mg mlÀ1 lysozyme solution
Summary
A powerful neutron diffraction technique for investigating protein chemistry, has elicited considerable interest among academics and pharmaceutical companies. Only 0.1% of the macromolecular structures deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) were determined using neutron diffraction (as of June, 2020) This is mainly because the neutron diffraction process requires much larger cubic crystals ($1 mm3) than X-ray diffraction. We developed a method for estimating the optimum experimental conditions for the growth of a large crystal using a certain volume of sample solution This method uses a combination of a few preliminary micro-batch experiments and a numerical model. The micro-batch experiments provide the empirical parameters for the model, and differential equations based on these parameters help to estimate the ideal conditions for the growth of a large single crystal in a certain sample volume. Microsoft Excel 2016 was used for the data analysis and all graphs
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