In this work we report on the violation of the succession of crystal growth modes as a function of supersaturation [1]. Typically, a crystal transitions from spiral growth to two-dimensional (2D) nucleation mediated growth with increasing supersaturation. In the case of monoclinic xylanase crystals we observed, using LCM-DIM, strong lattice discontinuities protruding the surface from which 2D and/or spiral hillocks originate. The complex nature of such a step source yields nonequidistant step trains and on some occasions even macrosteps demonstrating that the xylanase crystals grown under these conditions are far from perfect and do not behave like the quasi flawless model systems such as high purity lysozyme and glucose isomerase crystals.

More importantly, these defect-prone monoclinic xylanase crystals also constitute an exception to this well-maintained rule. This is the result of a unique interplay between the dominating layer generation mechanism and the subsequent occurrence and propagation of large numbers of lattice discontinuities. The defect density becomes so high that, given enough time, a fully developed, crystal-wide network of interlinked stacking faults is generated. This network effectively abolishes any advancement of steps emanating from the sole step source, being a spiral dislocation. The crystals manage to recover from this growth cessation by switching over to a secondary layer generating mechanism at lower supersaturation, that is, 2D nucleation. Several questions still remain open and need to be answered to fully elucidate the events observed on the monoclinic xylanase crystals. To what extent do these lattice discontinuities have an impact on the X-ray diffraction quality? The fact that high resolution structures of xylanase from Trichoderma are present in the protein databank (e.g., PDB-id: 2DFB [2]) suggests that these defects are not necessarily an intrinsic part of xylanase’s growth modes. But remarkably, all high resolution structures were obtained from the orthorhombic polymorph; no structures obtained from the mononclinic polymorph are reported. Taking into account the observations done in this work, it seems reasonable to assume that the high density of defects is related to a specific space group, monoclinic in this case, and does significantly affect the diffraction properties.

This observation demonstrate that by simply departing from well-established purified protein model systems, one can obtain unconventional and highly complex protein crystals with nontrivial growth mechanisms which could help us to understand the growth or (non-growth) on non-model proteins, still a major setback in structural biology.

[1]. M. Sleutel, Van Driessche A.E.S., Maes D. Cryst. Growth Des. 2012, 12, 2986-2993.

[2]. Watanabe, N.; Akiba, T.; Kanai, R.; Harata, K. Acta Crystallogr., Sect. D 2006, 62, 784−792.

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