The structure solution of T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9 µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotidecontrolled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

Summary In vitro allergy diagnostics are currently based on the detection of specific IgE binding on intact allergens or a mixture thereof. This approach has drawbacks as it may yield false‐negative and/or false‐positive results. Thus, we reviewed the impact of known B‐cell epitopes of food allergens to predict transience or persistence, tolerance or allergy and the severity of an allergic reaction and to examine new epitope mapping strategies meant to improve serum‐based allergy diagnostics. Recent epitope mapping approaches have been worthwhile in epitope identification and may increase the specificity of allergy diagnostics by using epitopes predominately recognized by allergic patients in some cases. However, these approaches did not lead to discrimination between clinically relevant and irrelevant epitopes so far, since the polyclonal serum IgE‐binding epitope spectrum seems to be too individual, independent of the disease status of the patients. New epitope mapping strategies are necessary to overcome these obstacles. The use of patient‐derived monoclonal antibodies instead of patient sera for functional characterization of clinically relevant and irrelevant epitope combinations, distinguished by their ability to induce degranulation, might be a promising approach to gain more insight into the allergic reaction and to improve serum‐based allergy diagnostics.