Microcrystal electron diffraction (MicroED) is a structural technique that leverages the strong interactions of matter with electrons. MicroED requires crystalline material, though material unsuitable for X-ray analysis is often permissible. A transmission electron microscope is used to collect diffraction data, which can be processed by standard crystallographic programs, allowing for rapid structure determination. The NIS MicroED workflow provides high resolution structures for challenging small molecule, peptide and macromolecular samples when starting material is extremely limited or when samples exhibit poor crystallization behavior. With MicroED, crystals that are one billionth the size needed for X-ray crystallography can be used to yield high quality data.
NIS has developed robust commercial services for small molecule microED studies to support a variety of industrial chemistry workflows. This service launch follows the successful determination of crystal structures for more than 20 small molecules during our alpha testing program. NIS' refinement protocols consistently produce low R-factors with goodness of fit (GooF) value close to 1 for samples ranging between 100 and 1000 Da.
Our small molecule microED services have been designed in a modular way to ensure maximum flexibility for clients to leverage internal expertise in conjunction with NIS' capabilities. NIS can perform all steps from sample preparation to the generation of publication-quality final structures. For clients with in-house experience in small molecule X-ray structure determination, data-acquisition only programs are also available. Learn more about our access programs through our Working With Us resource page.
Samples are initially transferred to a TEM grid in a manner that minimizes damage or possible changes to their properties. These processes must consider the possible effects of exposing samples to air, heat, light; dilution/resuspension in incompatible buffers/solvents or possible mechanical damage incurred during handling. Any of these conditions could affect both the amount of crystalline material on the grid and/or the quality of the electron diffraction that can be obtained.
We have implemented an initial workflow for microED data collection within our automated data acquisition framework.
Potential microcrystals are identified in low magnification TEM images. For verified crystals, a series of continuous-rotation, electron-diffraction datasets are collected from single crystals. A minimum of 20 datasets per sample are acquired. All microED data is collected on our Glacios microscopes equipped with CetaD cameras.
Processing microED diffraction data is similar to methods employed in traditional X-ray crystallography workflows. For clients processing the resulting microED data themselves, all necessary experimental parameters are provided. These can be input into the client's software of choice and assistance is available for certain software packages.
Depending on the characteristics of the small molecule and its crystal form, phasing may be carried out by ab initio and/or molecular replacement methods. Initial models are constructed and improved to generate a minimally refined structure. At the end of this phase, all atoms have been assigned and assessed for consistency with regard to the proposed 2D structure provided by the client. Hydrogens are added and preliminary ADP refinement is performed.
For projects in need of full refinement by NIS to address specific issues or for publication and/or regulatory filings, this optional second phase can be requested. Specific refinement issues may include more proper handling of crystal twinning, more rigorous analysis of potential space groups, handling severe crystal anisotropy or radiation damage, modeling structure disorder and rigorous investigation of areas of interest to the client, such as sites of proton transfer. This results in a fully validated structure with professional atom numbering and publication quality images.
NIS has defined the development of microED workflows from sample preparation through phasing as high priority project for 2020/2021. We are currently engaging with several industrial, academic and vendor collaborators to innovate in this space. Please contact usif you are interested in discussing use of this technique for one of your projects.
Get to know our team
Speak with our experts about using MicroED to support your drug discovery efforts.