X-ray protein crystallography has predominantly centred on growing large (> 5 µm) single crystals for structural analysis. While fragments of these large-grown, as well as small-grown crystals have been utilized in electron diffraction (3D ED/MicroED), a universal method for protein crystallisation tailored to sub-micrometre thickness is missing. In this study, we present a method wherein rapid mixing and fragmentation of initial crystals yield well-diffracting nanocrystals in seconds or minutes. In suitable space groups, these nanocrystals can form thin plates, providing ideal samples for (3D ED/MicroED) without requiring focused ion beam milling. The large number of suitably sized crystals also allows for the preparation of densely populated grids suitable for serial ED. We further introduce a streamlined manual grid preparation protocol using polysorbate 20 as a wetting agent, eliminating the need for glow discharging or plunge freezers. By applying these methods, micro- to nano-sized crystals of four different protein samples were generated and the resolution of each sample reached beyond 1.5 Å. Our high-resolution diffraction data demonstrate that slow and undisturbed growth is not necessary for obtaining well-diffracting protein crystals. This study offers a general and feasible sample preparation approach that enables high-resolution protein structure determination by electron diffraction.

Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (microED), is an emerging method for determining structures from submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single 3D ED/microED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of 3D ED/microED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based pipeline with a graphical user interface for automated real-time and offline batch 3D ED/microED data processing (AutoLEI). We demonstrate the functionality and applications of the pipeline through four examples, using both offline and real-time data processing capabilities. The samples include small organic molecules, metal–organic frameworks (MOFs) and proteins, showcasing the versatility and efficiency of AutoLEI in various applications.

Determining macromolecular structures is crucial for understanding biological mechanisms and advancing drug discovery. Recent advancements have highlighted the potential of electron diffraction using continuous sample rotation (MicroED) for resolving these structures from sub-micrometre-sized crystals. However, the achievable data quality of MicroED on proteins is limited by radiation damage of the crystal during collection. Serial electron diffraction (SerialED) avoids this issue by merging single-shot diffraction patterns from thousands of crystals. Despite its potential, the widespread use of SerialED has been limited by the complexity and rarity of the required equipment to acquire these single shot data. Here, we show a new continuous SerialED protocol that is simple, robust and universally accessible. It quickly and efficiently collects all diffraction data from an area without the need for prior crystal identification and enables structure determination of proteins at atomic resolution (0.83 Å) on widely available hardware. Furthermore, we found this technique to produce virtually radiation damage free structures, allowing us to measure radiation sensitive local chemical details and investigate protein-ligand interactions to state-of-the-art accuracy. Continuous SerialED significantly enhances the applicability of ED in structural biology by providing convenient, fast, and high-resolution data collection with minimal radiation damage. These advancements position continuous SerialED as a transformative tool in structural biology by providing high-quality protein structures using tools already available to scientists. We anticipate our protocol to enable a wide range of studies that require high quality diffraction data of radiation sensitive materials.

Understanding protein-ligand interactions is a key step for small-molecule drug discovery.  Conventional single-crystal X-ray diffraction provides accurate and high-throughput structural analysis. However, it is limited by the requirement for large crystals. Electron diffraction methods, such as microcrystal electron diffraction (MicroED/3DED) and serial electron diffraction (SerialED), offer ideal alternatives for structure determination of small protein crystals. Here, we present a workflow combining real-time MicroED and continuous SerialED (c-SerialED) to enable fast and accurate ligand binding analysis from microcrystals. By using the real-world cancer target human MutT homolog 1 (MTH1), we performed ligand soaking with four compounds of varying binding affinities. Each soaking event was completed within 3 seconds. Real-time MicroED analysis enabled confirmation of ligand binding within 25–50 minutes. High-resolution (1.7 Å) structures of the protein–ligand complexes were obtained through subsequent c-SerialED data collection. This workflow demonstrates the potential of using electron crystallography for structure-based drug discovery.