Vasireddy Purna Chandra Rao, Low-Beer Timothy, Spoth Katherine A, Acehan Devrim, Crawley Matthew R, Martynowycz Michael W
Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14203.
UB Hauptman-Woodward Institute, University at Buffalo, The State University of New York, Buffalo, NY 14203.
bioRxiv. 2025 Jul 5:2025.07.03.663097. doi: 10.1101/2025.07.03.663097.
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 Å resolution with an overall correlation coefficient of over 99% and solved the structure 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 Å) determined from spontaneously formed protein nanocrystals and is the first structure of crambin solved by electron diffraction. Our workflow demonstrates that complex biological matrices can be mined directly for sub-ångström protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets.
在纯化种子蛋白胰凝乳蛋白酶抑制剂（crambin）时，我们发现，在简单的乙醇纯化液滴干燥过程中会自发形成纯蛋白纳米晶体针状物。与传统晶体学不同，这些针状物用X射线衍射效果不佳，但事实证明它们非常适合微晶电子衍射（MicroED）。通过合并来自58个此类纳米晶体的数据，我们获得了分辨率为0.85 Å的衍射图，总体相关系数超过99%，并使用一个五残基螺旋片段启动密度修正来解析结构。所得图谱质量优异，能够实现全自动模型构建并分辨出单个氢原子。这项工作代表了从自发形成的蛋白质纳米晶体中确定的最高分辨率蛋白质结构（0.85 Å），并且是通过电子衍射解析的胰凝乳蛋白酶抑制剂的首个结构。我们的工作流程表明，可以直接从复杂的生物基质中挖掘亚埃级蛋白质结构，建立一条从原始生物质到以前难以处理的目标的原子级模型的实用且可扩展的流程。
