# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 4 The (Facultative?) Role of Accessory Proteins  
Crystallization of _Bt_ toxins sometimes requires the involvement of additional proteins. They have been coined with many different names including 'chaperone-like proteins' [44],'molecular chaperones' [45], 'crystallization proteins' [19], 'helper proteins' [46; 47], or even 'accessory proteins' [48]. Obviously, this plethora of appellations has been more misleading than informative. Indeed, the fact that their absence (sometimes) leads to the absence of a crystal does not necessarily mean that they are directly and actively involved in the crystallization process. They may act at different levels--e.g., they can stabilize the protein and/or protein-protein interactions, help protein folding or increase its concentration--which both help to achieve the required conditions for crystallization without actually directly facilitating or participating in crystal contacts. Therefore, one should be careful with the terminology employed. In this part and elsewhere in the article, we use the term 'accessory proteins' which encompasses all the proteins directly or indirectly implicated in _Bt_ toxin crystallization as it provides no a priori information on the mechanism involved, which is sometimes unknown or unclear. These accessory proteins are generally organized in an operon together with _Bt_ toxin gene(s) [18; 19]. The operons of several 55-65 kDa toxins have, downstream of the toxin-coding gene, a second open reading frame (ORF2) with high sequence homology to the 'crystallization domain' of the 130-140 kDa toxins (discussed in Section 3). A series of experiments fusing the ORF2 of Cry19A with the Cry19A toxin or with the N-terminal core toxin domain of Cry1C both led to the formation of inclusions [49]. This suggests that ORF2 might play the same role as the 'crystallization domain' of 130-140 kDa toxins through a similar, albeit still poorly understood, mechanism [18; 20]. The efficiency and universality of such a domain/ORF2 might explain why it is so widespread in many different _Bt_ subspecies for a number of different toxins within the Cry structural group [3; 18]. Another kind of accessory protein called P29 (due to its 29 kDa size) is found in the operon of Cry2A toxin and contains a 15-residues motif repeated eleven times [50]. It has been hypothesized that it would form a framework favoring the nucleation of Cry2 Aa toxins as they are synthesized, driving their assembly into characteristic cuboidal crystals [51; 52]. While repeat sequences have been identified in the C-terminal region of other toxins, their exact role in crystallization is still unknown [18]. P20, a protein of 20 kDa, is encoded by the third ORF in the Cry11 Aa operon from _Bt_ subsp. _israelensis_ (_Bit_) and it has been shown to be mandatory for the crystallization of Cyt1 Aa [53], whose coding gene is located in the neighboring operon upstream Cry11 Aa's one in the pBToxis megaplasmid [54]. The structural modelling of P20 revealed that it exhibits a high structural homology with the activated form of Cyt1 Aa [55]. Knowing that P20 forms a transient complex with the nascent Cyt1 Aa peptide chain [56], one could hypothesize that it offers a scaffold for the setting of the domain-swapping of the Cyt1 Aa N-terminal domain [11], in line with the \"heritable template\" hypothesis [57]. Interestingly, _Bt_ subsp. _medellin_ possesses a homolog of P20--namely P21--located in 5\({}^{\prime}\) of a homolog of Cyt1 Aa--namely Cyt1 Ab--with whom they share 75% and 86% identity, respectively [58]. This would further argue in favor of a co-evolution between these accessory proteins and their Cyt1 counterpart. Interestingly, while the non-cytolytic Cyt2 Ba can crystallize in vivo and in vitro through a similar domain-swapping strategy, it does not involve any P20-like protein [41].