Serial synchrotron crystallography in drug discovery - using on-chip co-crystallization on silicon nitride membranes

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Examensarbete för masterexamen
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2022
Författare
Wehlander, Gabrielle
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Background: Serial synchrotron crystallography (SSX) has reached an increased popularity over the last years, predominantly thanks to its potential to derive room-temperature (RT) structures of proteins, with a low risk of causing radiation damages. Structure determination of ligand bound protein complexes is of major importance in today’s drug development research, of which structure-based design is an important part. However, efficient methods for producing such samples, suitable for SSX, has not yet been established. It is therefore of uttermost importance to evaluate methods to efficiently produce protein-ligand complex crystals, to allow for high-scale implementation of SSX in the drug industry. One such method with promising potential is to perform on-chip co-crystallization of protein-ligand bound complexes on fixed-target sample delivery supports. By introducing the ligand by drying it on the supports further opens the possibilities of automating parts of the sample production. Aim: The aim of the project was to i) evaluate the potential of performing on-chip crystallization on silicon nitride membranes, used as fixed-target supports at the MAX IV Laboratory, and ii) to evaluate the capability of two different co-crystallization techniques combined with on-chip crystallization. Method: The protein soluble epoxide hydrolase was used as a proof-of-concept protein to evaluate on-chip co-crystallization on the silicon nitride membranes. The two co-crystallization techniques tested were i) to incubate protein and ligand in solution prior to crystallization, and ii) to dry ligand on the membranes, followed by adding protein crystallization drops. The capability of the two techniques was validated with RT-SSX at BioMAX, MAX IV. Results: On-chip co-crystallization on the silicon nitride membranes was shown to be both possible and to generate well-diffracting crystals for RT-SSX. Structure analysis of the RT-SSX collected data showed that both co-crystallization techniques generated ligand bound protein complexes. Discussion: The successful application of on-chip co-crystallization on the membranes makes it of highest interest to further evaluate the method using other proteins and ligands, to evaluate the possibility of implementing the method in the drug industry.
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Room-temperature serial synchrotron crystallography, fixed-target sample delivery, on-chip crystallization, co-crystallization, soluble epoxide hydrolase, silicon nitride membranes
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