Microfluidic Protein Flow Crystallization Using Engineered Nucleation Features for Serial and Traditional Crystallography

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PROJECT SUMMARY DeNovX creates innovative platform products that improve crystallization. Phase I seeks to improve the crystallization and sample handling efficiencies of high impact infectious disease related proteins by incorporating engineered nucleation features (ENFs) into microfluidic flow crystallization chips compatible with single crystal and serial crystallography using synchrotron X-ray and free electron laser (XFEL) lightsources. Crystal nucleation of proteins is challenging with the best workflows still averaging ≥ 80-85% failure rates. DeNovX’s ENFs reduce the thermodynamic and kinetic barriers to crystal nucleation, and combining ENFs with microfluidic flow crystallization benefits structural biology by producing more protein crystals for fixed and flowing sample targetry in the emerging “diffract before destroying” strategies with high brilliance X-rays and by more efficiently using the protein resources. X-ray crystallography remains a benchmark technique by providing unparalleled atomic resolution data that serve as models for cryo-EM and NMR structures, and benefits to Public Health derive from an accelerated and expanded understanding of disease genesis, progression, and therapy. Specific Aim 1 - Define microfluidic protein flow crystallization chip formats and incorporate ENFs. Using as benchmarks select carbohydrate active enzyme (CAzyme), ꞵ-lactamase, and SARS-CoV-2 (e.g., Nsp15, Mpro, PLpro) proteins, collect replicate (n ≥ 6) crystallization hit percentage, crystal yield, and onset time data with 12 unique ENFs vs. control surfaces for the polydimethylsiloxane (PDMS)/glass microfluidic materials of construction using microbatch crystallization. Identify the top four ENFs showing reproducible improvements of ≥ 10% increase in crystallization hits, ≥ 20% increase in the quantity of crystals generated, or ≥ 15% reduction in crystallization onset times vs. controls. Specific Aim 2 - Design a microfluidic protein flow crystallization platform incorporating ENFs that can produce and transport: (a) 1-50 µm crystals for fixed target meshes and flowing sample microjet injection for serial femtosecond crystallography using XFELs, and (b) 50-100 µm protein crystals for traditional single crystal diffraction. Assemble two functional PDMS/glass α-prototypes with ≥ 3 fluid addition points for manipulation of crystallization conditions, establish hydrodynamic conditions for operation, and demonstrate efficient transport of 1-50 µm and 50-100 µm protein crystals with ≤ 25% average change in droplet size (may affect crystal size). Specific Aim 3 - For protein microfluidic flow crystallization using select ENFs and benchmark proteins (CAzymes, ꞵ-lactamases, SARS-CoV-2), demonstrate reproducible (n ≥ 6) improvements of ≥ 20% increase in the quantity of crystals generated, ≥ 20% reduction in crystallization onset time, or ≥ 20% narrowing of crystal size distribution vs. controls. Confirm using synchrotron X-rays that structure quality metrics (e.g., resolution, R, etc.) of protein crystals are within ± 3 esds of PDB benchmarks. It is expected that microfluidic protein flow crystallization will efficiently produce diffraction quality crystals to enhance the quality and quantity of protein structure determination studies.