Protein crystallization for structure determination often relies on seeding to bypass the stochastic nucleation step and produce well-ordered crystals, yet traditional seed‐preparation methods—such as manually crushing macroscopic crystals, sonicating drops or relying on harsh mechanical disruption—yield highly heterogeneous seed populations, include amorphous debris or over-fragmented fragments, and lack quantitative control over active nuclei. Distinguishing beneficial seeds from deleterious particles is laborious, and small variations in seed size or concentration can drastically alter crystallization outcomes, leading to poor reproducibility and making it difficult to optimize nucleation density. Attempts to standardize seed stocks through filtration, dilution series or centrifugation often incur sample loss, damage fragile crystals and fail to deliver uniform, tunable seed distributions, thus limiting the applicability of microseeding to proteins that form only micro- or nanometer precursors.
Description
The technology provides a simple, reproducible method and accompanying kit for generating protein nanoseeds by mechanically fragmenting protein nanocrystal aggregates with sub-millimeter beads (0.05–5 mm; glass, metal, ceramic or PTFE) in a microcentrifuge tube, producing a quantifiable slurry of uniform seeds whose size distribution (≈0.5 µm to >300 µm) can be precisely tuned by bead diameter and agitation time (up to 30 s). Aggregates are pre-selected by brightfield or UV tryptophan fluorescence microscopy and confirmed by TEM before seeding, and the resulting nanoseeds—unlike traditional microseeding approaches that rely on manual crushing or bulk seed stocks—deliver homogeneous, high-quality crystals even from previously intractable or optically invisible nanocrystals, thereby improving crystal growth reproducibility, resolution and mosaicity.
Applications
• Structural biology and drug discovery: enables generation of high-quality crystals for challenging protein targets, accelerating X-ray crystallography workflows in pharmaceutical and biotechnology R&D.
• High-throughput crystallization: improves success rates and reproducibility in microseeding for contract research organizations and core facilities offering crystallization screening
• Protein crystallization consumable kits: provides researchers with ready-to-use bead-and-tube kits for reliable nanoseed generation, simplifying setup and standardizing seed production in crystallography labs.
• Biopharmaceutical development and quality control: facilitates precise structural characterization of biologics and enzymes through improved crystal quality and size homogeneity, supporting formulation development and batch consistency testing.
Advantages
• Improved seed homogeneity
• Broader applicability to recalcitrant proteins
• Enhanced crystal quality
• Cost-effective workflow
• Rapid seed generation
• Controlled crystal catalogues
• Integration with UV screening
• Protein-friendly agitation
IP Status
https://patents.google.com/patent/US20150251106A1
