Vasireddy, P. C. R.; Low-Beer, T.; Spoth, K. A.; Acehan, D.; Crawley, M. R.; Martynowycz, M. W.

While purifying the seed protein crambin, we discovered that needles of pure protein nanocrystals formed spontaneously during the drying of a simple ethanolic purification drop. Contrary to traditional crystallography, these needles diffracted poorly using X-rays yet proved to be exceptionally well-suited for microcrystal electron diffraction (MicroED). By merging data from 58 such nanocrystals, we obtained diffraction to 0.85 A resolution with an overall correlation coefficient of over 99% and solved the structure ab initio using a five-residue helical fragment to initiate density modification. The resulting map was of exceptional quality, enabling fully automated model building and resolving individual hydrogen atoms. This work represents the highest-resolution protein structure (0.85 A) determined from spontaneously formed protein nanocrystals and is the first ab initio structure of crambin solved by electron diffraction. Our workflow demonstrates that complex biological matrices can be mined directly for sub-angstrom protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets.