# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 3 Toxins Evolved to Be Crystallization-Prone  
Expressing a very high quantity of proteins is not sufficient to make an ordered assembly of protein leading to a crystal; instead, it generally leads to inclusion bodies with little intrinsic organization [36]. The _Bt_ crystal properties--i.e., stability in aqueous environment, solubility restricted to high pHs only and protease-resistant core protein--are driven by the toxin itself and its crystallization properties, which imposes the toxin's structural characteristics. The toxin Cry3 Aa, for example, crystallizes through a checkerboard-like packing into crystals with high solvent content (Figure 3A) [37]. Its dissolution is restricted to high pH by four intermolecular salt bridges located at contacts with three different neighboring molecules [38]. Interestingly, Cry3 Aa protoxin is able to crystallize both in vivo and in vitro and it exhibits the same crystal-packing interfaces, highlighting that the crystallization process of this toxin is essentially, if not exclusively, driven by intrinsic protoxin characteristics [37; 38; 39]. However, this is not a feature common to all _Bt_ toxins as some are unable to crystallize outside the cell in their protoxin form, such as the 27 kDa Cyt1 Aa (Figure 3B). Indeed, only the protease-resistant core protein of Cyt1 Aa could be recrystallized in vitro after the N-terminal propeptide had been proteolytically removed from the toxin [17]. The Cyt1 Aa protoxin structure directly solved from in vivo crystals allowed to elucidate the reason behind this phenomenon [11]. During the sporulation of the _Bt_ cell, crystallization of Cyt1 Aa is initiated by a domain-swapping of the N-terminal propeptide of two nascent Cyt1 Aa monomers, leading to the formation of a domain-swapped dimer. This interface drives the crystallization of Cyt1 Aa and it has the largest buried surface area (BSA) in the crystals. The dissolution of Cyt1 Aa crystals relies on the dissociation of this dimer by electrostatic repulsion between residues around the domain swapping area [11]. Recrystallizing the protoxin after crystal dissolution would require the swapping of the N-terminal propeptide to be reconstituted, which corresponds to a high free-energy barrier hardly reachable in vitro [40]. This could explain why only activated Cyt1 Aa toxins devoid of N-terminal propeptides could be recrystallized in vitro and why the crystal packing differs between in vivo crystals of protoxin and in vitro crystals of toxin [11; 17]. Intrigu  
Figure 3: Different _Bt_ toxins with different structures exhibit different crystallization strategies to achieve similar crystal features. Here two examples are shown of toxins structures solved directly from crystals grown in _Bt_. (**A**) The stacking of the three-domain Cry3 Aa protoxins allows generating solvent channels of 3–5 nm width that traverse the crystal cell along the \(c\) axis. The same crystal packing is observed in in vivo [37] and in in vitro crystals [38]. (**B**) In vivo crystallization of Cyt1 Aa is essentially driven by its homodimerization through the domain-swapping of the N-terminal propeptides [11], which differs from the crystallization of the activated Cyt1 Aa toxin in vitro [17]. 3D structures of Cyt1 Aa (PDB accession number: 6T14 [11]) and Cry3 Aa (PDB: 4 QX0 [37]) protoxins are represented in ‘cartoon’ mode and colored from blue (N-terminal) to red (C-terminal) using PyMOL Molecular Graphics System 2.1.1.  
ingly, the homologous non-cytolytic Cyt2 Ba protoxin, which exhibits a similar 3D structure and shares 33% of sequence identity with Cyt1 Aa, could crystallize in vitro with a crystal packing driven by a N-terminal domain-swapping similar to Cyt1 Aa in vivo crystals [41]. This might be linked to the involvement of a chaperone for Cyt1 Aa crystallization, which is absent for Cyt2 Ba (see Section 4 for further discussion). _Bt_ toxins of 130-140 kDa, comprising the anti-lepidopteran Cry1 groups of toxins, rely on yet another crystallization mechanism [18].