UCLA researchers from the Department of Chemistry and Biochemistry have developed a novel method to obtain atomic and molecular structures from digitally defined regions of a sample.
BACKGROUND:
Crystallography and diffraction experiments allow insightful looks into the atomic structure of materials. The usefulness of crystallography is vast, ranging in the life sciences from helping to understand the design of drugs to target specific proteins for various therapeutic purposes, to the physical sciences in helping to determine multiple material properties. Unfortunately, there are certain impediments to the use of crystallography, like the need to grow sufficiently large crystals for accurate structure determination. While recent advancements have led to methods that allow tiny crystals to be characterized, irregularities in crystalline samples can lead to challenges in the detection or interpretation of diffraction signals, currently hampering these techniques. A multitude of approaches has been developed to overcome structure irregularities, such as serial crystallography. While this technique can characterize small crystal sizes with irregularities, it requires a significant number of crystals. Other techniques, like selected area electron diffraction, allow researchers to use physical apertures that define areas of interest within a crystal but require sample or aperture adjustments when choosing a different region of interest. Therefore, there currently exists a need for new crystallography techniques that offer the ability to characterize small crystals that may have structural irregularities, those that are part of a mixture of structures or have different structural packings, while also providing accurate high-resolution structures.
INNOVATION:
UCLA researchers have developed a novel scanning nanobeam electron-diffraction tomography (NanoEDT) method to define structures from digitally defined regions of interest. By utilizing a direct electron detector, the researchers can record thousands of diffraction patterns over multiple crystal orientations: overcoming previous crystal size limitations in structure elucidation. Each diffraction pattern is assigned a specific location on a single nanocrystal with axial, lateral, and angular coordinates. This method creates a four-dimensional dataset of the crystal, allowing the ability to digitally extract intensities from any desired region of the scan in real or diffraction space, exclusive of all other scanned points. This is achieved in a semi-automated fashion through the use of software interfaces. In this methodology, irregularities in the crystal structure can be avoided, while also providing high-resolution in structure determination. As a proof of principle for this technique, the researchers determined the atomic structure of a peptide from specific regions of nanocrystals, precisely outlined after data collection. This method eliminates the need for a pre-defined diffraction aperture during data collection and opens a new realm of possibilities from arbitrarily defined regions of crystalline samples.
APPLICATIONS:
• Structural determination of nanocrystalline samples, exceptionally heterogeneous or polycrystalline nano assemblies
• Can address unresolved questions about sources of error in electron diffraction and yield structures with improved accuracy
ADVANTAGES:
• Allows users to select nanoscale areas of a crystal for structural determination and refinement
• Data correlates well to data collected by conventional MicroED or powder X-ray methods
DEVELOPMENT-TO-DATE:
Researchers have defined a size-residue segment from the OsPYL/RCAR5 protein and determined that the data correlated well with data from a MicroED experiment.
RELATED MATERIALS:
Gallagher-Jones, M. et al. Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp. Nature Structural & Molecular Biology 25, 131–134 (2018).
Gallagher-Jones, M. et al. Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction. Communications Biology 2, 1–8 (2019)
Jones, C. G. et al. The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination. ACS Cent. Sci. 4, 1587–1592 (2018).