# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 5 Crystallization in Bt Is a Finely-Tuned Multifactorial Process  
Each of the three factors presented above (i.e., the finely-regulated production of high quantities of toxins, their crystallization proneness, and the facultative presence of accessory proteins) rarely solely drive toxin crystallization [18; 19; 20]. They are highly toxin-dependent, meaning that they might be neither universal nor mandatory, and while they can exhibit complementary functions, they are not mutually exclusive. For example, [PERSON] et al. demonstrated that crystals of Cry1 toxins obtained in vivo by _Bt_ and in vitro after denaturing treatment with urea both formed bipyramidal crystals, indicating that the shape and size of crystals were driven by the toxin itself [61]. However, the pH at which they dissolved strongly differed, i.e., 9.5-10.5 vs. 12 for in vivo and in vitro crystals, respectively, which supports that some key protein-protein interactions--notably involving disulfide bonds--that drive crystal dissolution can be stably established only within the bacterial cell [19]. This contrasts with Cry3 Aa that can crystallize in vivo and in vitro while conserving similar features [37; 38] and with Cyt1 Aa for which the protoxin is unable to recrystallize in vitro [17], notably due to contrasting crystal packing strategies between the protoxin and the toxin [11]. As discussed above (see Section 2), Cry3 Aa production is controlled by a sigma factor (\(\sigma^{\text{A}}\)) active at the vegetative stage and its mRNA is stabilized by the STAB-SD sequence [34]. It was however shown that the sporulation promoters of Cyt1 Aa (\(\sigma^{\text{E}}\) and \(\sigma^{\text{K}}\)[22]) were sufficient to compensate for the absence of both \(\sigma^{\text{A}}\) and STAB-SD to produce similarly shaped and sized crystals of Cry3 Aa [62]. However, when Cyt1 Aa promoters were combined with STAB-SD, this allowed a significant increase in yield of Cry3 Aa produced and the size of the crystal, but this came with high fitness costs resulting in a reduced number of spores produced [62]. STAB-SD has been used to increase the yield and subsequent crystal size of different toxins with a large variation in the outcome, with an order of magnitude between the lowest (e.g., for Cry2 Aa [49; 63] and Cry11 Aa [63]) and the highest increase (e.g., Cry11 Ba [64], Cry19 Aa [49], Mpp60 Aa and Mpp60 Ba--formerly known as Cry60 Aa and Cry60 Ba, respectively [65]). This exemplifies how different factors affecting similar parameters (here, the number of proteins produced per transcript by increasing its number and/or by stabilizing it) can complement themselves and that additional toxin-dependent factors seem to regulate the relative contribution of each factor, notably to balance the energetic needs for the concomitant formation of the crystal and the spore. Determining the role of each factor is therefore necessary but insufficient to understand the whole crystallization process. Notably, more crystallographic studies directly using crystals grown in \(Bt\) are required to widen our understanding of the underlying crystallization mechanisms, in addition to providing key information on the toxin's mode of action. The (R)Evolution of Structural Biology Allows the Unraveling of Key Steps in the Crystallization Pathways of \(Bt\) Toxins  
Determining the structure of a protein is a key milestone in deepening the study and understanding of its function. Among all the methods available in structural biology, macromolecular crystallography (MX) remains the favored one for determining the structure of proteins at high resolution (Figure 4) [66]. It is notably due to the eased access to finely tuned and reliable MX beamlines at synchrotron sources that this technique has been used to solve >90% of the structures present in the Protein Data Bank (PDB).