Richards, L. S. | bioRxiv Science

bioRxiv2023

Cryo-EM Structure of a Human LECT2 Amyloid Fibril Reveals a Network of Polar Ladders at its Core

ALECT2 is a type of systemic amyloidosis caused by deposition of the leukocyte cell-derived chemotaxin-2 (LECT2) protein in the form of fibrils. In ALECT2, LECT2 fibril deposits can be found in the glomerulus, resulting in renal failure. Affected patients lack effective treatment options outside of renal transplant or dialysis. While the structure of LECT2 in its globular form has been determined by X-ray crystallography, structures of LECT2 amyloid fibrils remain unknown. Using single particle cryo-EM, we now find that human LECT2 forms robust twisting fibrils with canonical amyloid features. At their core, LECT2 fibrils contain two mating protofilaments, the ordered core of each protofilament spans residues 55-75 of the LECT2 sequence. The overall geometry of the LECT2 fibril displays features in line with other pathogenic amyloids. Its core is tightly packed and stabilized by a network of hydrophobic contacts and hydrogen-bonded uncharged polar residues, while its outer surface displays several charged residues. The robustness of LECT2 fibril cores is illustrated by their limited dissolution in 3M urea and their persistence after treatment with proteinase K. As such, the LECT2 fibril structure presents a potential new target for treatments against ALECT2.

biochemistry↗

bioRxiv2019

Atomic structures determined from digitally defined nanocrystalline regions

Nanocrystallography has transformed our ability to interrogate the atomic structures of proteins, peptides, organic molecules and materials. By probing atomic level details in ordered sub-10 nm regions of nanocrystals, approaches in scanning nanobeam electron diffraction extend the reach of nanocrystallography and mitigate the need for diffraction from large portions of one or more crystals. We now apply scanning nanobeam electron diffraction to determine atomic structures from digitally defined regions of beam-sensitive peptide nanocrystals. Using a direct electron detector, we record thousands of sparse diffraction patterns over multiple crystal orientations. We assign each pattern to a specific location on a single nanocrystal with axial, lateral and angular coordinates. This approach yields a collection of patterns that represent a tilt series across an angular wedge of reciprocal space: a scanning nanobeam diffraction tomogram. From this diffraction tomogram, we can digitally extract intensities from any desired region of a scan in real or diffraction space, exclusive of all other scanned points. Intensities from multiple regions of a crystal or from multiple crystals can be merged to increase data completeness and mitigate missing wedges. Merged intensities from digitally defined regions of two crystals of a segment from the OsPYL/RCAR5 protein produce fragment-based ab-initio solutions that can be refined to atomic resolution, analogous to structures determined by selected area electron diffraction. In allowing atomic structures to now be determined from digitally outlined regions of a nanocrystal, scanning nanobeam diffraction tomography breaks new ground in nanocrystallography.

biophysics↗