# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 5 Crystallization in Bt Is a Finely-Tuned Multifactorial Process
Although informative, they both offer a restricted view on the implication of a limited set of amino acids lacking most of the interactions that can drive the toxin function as those cannot be easily extrapolated from the protein amino acid sequence. Recent advances in crystallography methods allowed the unraveling of some hidden features driving the in vivo crystallization process of a few \(Bt\) toxins. A major breakthrough has been the introduction of serial crystallography--whereby diffraction data are collected from a myriad of still crystals instead of a single crystal oscillating in the X-ray beam--at X-ray free electron lasers, whose peak brillance is fifteen orders of magnitude higher than synchrotrons, resulting in the delivery of the same amount of photons in a \(\sim\)40 fs pulse, as the latter would produce in 1-10 ms [77]. This method is generally limited to a restricted number of proteins able to crystallize easily in vitro due to the high amount of protein needed for performing an experiment (from 0.05 to 1 gram, depending on the experimental goal). In the case of \(Bt\), its proneness to produce normalized, micro-meter sized crystals in laboratory settings, the stability of its crystals in water, and the availability of many procedures for crystal purification made it perfectly adapted for such an approach. This led to the determination of the structures of the full-length toxins Cry3 Aa [37] and Cyt1 Aa [11] from \(Bt\), but also of the binary toxin Tpp1 Aa/2 Aa (formerly BinAB) from _Lysinibacillus sphareicus_ produced in \(Bt\)[78]. This notably allowed confirming the moieties that support the crystallization-proneness of Cry3 Aa [37; 38] and the unveiling of the key role played by the N-terminal propeptide of Cyt1 Aa during its in vivo crystallization [11; 17]. The opening of new MX beamlines at operating XFELs and the will of such facilities to tend toward a more user-friendly platform hold promise that access to these facilities will become less competitive in the future, enabling more experiments to be performed [77]. Moreover, the success of serial crystallography at XFELs inspired investigators to import serial crystallography at synchrotrons (SSX) [79; 80; 81; 82; 83]. With the upgrade of synchrotrons to \"extremely brilliant sources\" (EBS, Figure 4), resulting in a \(10^{1}\)-\(10^{3}\) fold increase in flux at microfocus beamlines [84], and the installation at all synchrotron facilities of dedicatedserial crystallography beamlines, SSX experiments are on the edge of democratization, expanding the possibilities for \(Bt\) studies. In parallel to the development of crystallography, cryo-electron microscopy (cryo-EM) also experienced a revolution, pushing back the limits of resolution attainable (Figure 4) [85]. Using this technique, the structures of the secreted soluble Vip3 Aa and Vip3 Bc toxins were determined, providing key information into their activation, oligomerization, and membrane perforation processes [86; 87]. Hence, the forthcoming years could allow a significant number of structures of native \(Bt\) toxins to be solved, significantly expanding our understanding of the diversity of toxin crystallization strategies in \(Bt\).