Carboxylic acids in crystallization of macromolecules: learning from successful crystallization experiments

Abstract

The production of macromolecular crystals suitable for structural analysis is one of the most important and limiting steps in the structure determination process. Often, preliminary crystallization trials are performed using hundreds of empirically selected conditions. Carboxylic acids and/or their salts are one of the most popular components of these empirically derived crystallization conditions. Our findings indicate that almost 40 % of entries deposited to the Protein Data Bank (PDB) reporting crystallization conditions contain at least one carboxylic acid. In order to analyze the role of carboxylic acids in macromolecular crystallization, a large-scale analysis of the successful crystallization experiments reported to the PDB was performed. The PDB is currently the largest source of crystallization data, however it is not easily searchable. These complications are due to a combination of a free text format, which is used to capture information on the crystallization experiments, and the inconsistent naming of chemicals used in crystallization experiments. Despite these difficulties, our approach allows for the extraction of over 47,000 crystallization conditions from the PDB. Initially, the selected conditions were investigated to determine which carboxylic acids or their salts are most often present in crystallization solutions. From this group, selected sets of crystallization conditions were analyzed in detail, assessing parameters such as concentration, pH, and precipitant used. Our findings will lead to the design of new crystallization screens focused around carboxylic acids.

Structure of dihydrodipicolinate synthase from the commensal bacteriumBacteroides thetaiotaomicronat 2.1 Å resolution

“…Crystallization experiments were performed at room temperature using the sitting-drop vapor-diffusion method in 96-well sitting-drop plates (Molecular Dimensions, Alamonte Springs, Florida, USA). A solution of recombinant BtDapA was mixed with well solution from a custom carboxylic acid screen (Offermann et al, 2014). Crystals were grown in 0.1 M succinate pH 7.0, 0.1 M acetate, 0.8 M NaCl, 10 mM 5-aminoisophthalic acid, 15%(w/v) PEG 3350.…”

Tailored Suits Fit Better: Customized Protein Crystallization Screens

“…Besides the above-mentioned bias of sparse matrix screens, additional drawbacks come from the mode of use: usually, all the conditions in the screens are used, even when there is prior knowledge that a specific condition or factor is detrimental. In one of the surveys on crystallization conditions, Fazio et al 9,10 showed that the collection of successful compounds has changed in the past decade and that commercial screens sparsely cover some of these crystal yielding conditions. A similar conclusion was drawn by Gorrec 11 who showed that the 1440 crystallization conditions routinely used in the crystallization facility at MRC Laboratory of Molecular Biology (MRC-LMB, Cambridge, UK)) under-sample the chemical space.…”

“…In one of the surveys on crystallization conditions, Fazio et al , showed that the collection of successful compounds has changed in the past decade and that commercial screens sparsely cover some of these crystal yielding conditions. A similar conclusion was drawn by Gorrec who showed that the 1440 crystallization conditions routinely used in the crystallization facility at MRC Laboratory of Molecular Biology (MRC-LMB, Cambridge, UK)) under-sample the chemical space.…”

A drunken search in crystallization space

“…In the article by Luft and coworkers, the 'mini-phase diagram' approach to initial crystallization suggests using chemicals that have already been proven to be successful in protein crystallization at concentrations that have already been successful (Chan et al, 2013;Luft et al, 2014). Analyses of the most common chemicals used in crystallization have been performed before (Peat et al, 2005;Lu et al, 2012;Parker & Newstead, 2012;Offermann et al, 2014), and we wondered whether the conclusions from these earlier studies still hold today.…”