# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 5 Crystallization in Bt Is a Finely-Tuned Multifactorial Process
In synchronous, diffraction data are generally collected from a single macro-crystal (10-100 \(\upmu\)m) maintained in cryogenic conditions to collect a complete dataset by oscillation methods, yielding structures with mitigated radiation damage [67; 68]. Unfortunately, the submicrometer size of the crystals naturally produced by \(Bt\) has long hindered their study, as they were not amenable to structure determination by conventional oscillation methods at synchrotron sources. This is because the progression of X-ray-induced radiation damage is only delayed but not eliminated by flash-cooling. In practice, this means that the smaller the crystals, the more radiation sensitive they are, requiring more crystals to obtain a radiation damage-free dataset [69]. Figure 4: Participation of the three major structural methods in the determination of protein structures. (**A**) Yearly evolution of protein structure deposition on the RCSB Protein Data Bank (PDB, [70]). Key milestones are indicated below the graph. (**B**) Box plot representation of the resolution limit (in Angstrom (Å)) for X-ray crystallography and Cryo-EM. (**C**) Box plot representation of the protein size (in Dalton) is indicated for NMR, X-ray crystallography, and Cryo-EM. For the entire figure, data related to X-ray crystallography, Nuclear Magnetic Resonance (NMR), and cryo-electron microscopy (Cryo-EM) are indicated by blue, green, and orange colors, respectively. ESRF, European Synchrotron Radiation Facility (Grenoble, France). Data were retrieved from the RCSB database on 31 January 2021 ([[https://www.rcsb.org](https://www.rcsb.org)]([https://www.rcsb.org](https://www.rcsb.org))). Investigation of the link between structural features and \(Bt\) toxin function had to rely on direct and/or indirect approaches to deal with the \(Bt\) crystal size limitation. A direct approach consisted in the solubilization of the natural \(Bt\) toxin crystals in alkaline buffers before toxins were recrystallized in vitro onto bigger crystals compatible with MX beamlines of synchrotrons. Although a restricted number of toxins could recrystallize in their full-length, such as Cry3 Aa [38], Gpp34 Aa (Cry34 Ab) [12], Tpp35 Ab (Cry35 Ab) [12], and Cyt2 Ba [41], most \(Bt\) toxins are reluctant to recrystallization in vitro [4]. This can be attributed to the need of accessory proteins, of the specific intracellular environment and/or of particular toxin-toxin interactions driven by propeptides during the early stages of protein production [18; 19]. To circumvent this problem, the most adopted strategy consisted in mimicking the toxin activation step within the insect gut by proteolytically activating the full-length toxin in vitro to release the core toxin devoid of its N- and/or C-terminal parts. By doing so, several structures of activated \(Bt\) toxins were obtained, providing key information on the domains and amino acids involved in insect receptor recognition, oligomerization, and cell perforation, notably driving point-mutation strategies to improve toxin efficacy and host spectrum [71]. However, they offered a limited view of the mechanism of crystallization within the cell, as the crystal packing of activated toxins may differ from the packing of full-length toxins in \(Bt\) cells [11]. The absence of structures for most toxins prompted scientists from the field to embrace indirect strategies to investigate the implication of given amino acid(s) in the function of the toxins. Some used a mutation-based approach, which consists of the mutation of a selected set of amino acids considered as important, based on the sequence, on domain prediction and on homology of the protein sequence with other known \(Bt\) toxins. Then, the effect of these mutations on the toxin activity and capacity to form crystals can be observed [72; 73; 74]. Tridimensional homology modelling was also conducted to explore the interactions between toxin domains and potential insect receptors [55; 76; 57].