Biology subjects

Miller, R. J. D.

Publications and source records attributed to Miller, R. J. D..

4 recordsLinked to original sources

bioRxiv2025

Electron diffraction captures high-resolution structures from in vivo protein nanocrystals of Bacillus thurigiensis

Bacillus thuringiensis is one of the most widely used biopesticides worldwide. This is owing to the highly-specific pesticidal-proteins various strains produce in the form of nanocrystals. Structure determination from such crystals remains difficult because their small size makes them unsuitable for conventional X-ray crystallography. Here we explore two emerging (cryo-) electron diffraction techniques, namely Microcrystal electron diffraction and serial electron diffraction, as tools for studying the structures of these crystals. Using the mosquitocidal protein Cry11Aa as an example, we compare electron diffraction with state of the art results obtained with an X-ray free electron laser. Our work demonstrates that electron diffraction is a viable alternative for structure determination from such challenging crystals, matching previous results obtained with X-ray free electron lasers. We present a workflow based on readily available instrumentation enabling structure determination directly from the crystals grown in vivo, unperturbed by dissolution and therefore preserved in their native state.

biophysics↗

bioRxiv2021

Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging

Cataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, {gamma}S-crystallin ({gamma}S): one of the wild-type in a monomeric state, and five of deamidated {gamma}S variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single-to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of {gamma}S; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of {gamma}S-crystallin in the human lens. HighlightsO_LINovel structures of cataract-associated variants of human eye lens protein {gamma}S-crystallin reported C_LIO_LIIncreasing deamidation of {gamma}S-crystallin decreases stability and affects aggregation propensity C_LIO_LIOverall fold of {gamma}S-crystallin maintained among deamidated and disulfide-bonded variants C_LIO_LIDeamidated {gamma}S variants form disulfide bonds more rapidly than wild-type {gamma}S C_LIO_LIPotential functional advantage of disulfide bonding in the CXCXC motif proposed C_LI

biochemistry↗

bioRxiv2020

Serial femtosecond and serial synchrotron crystallography yield data of equivalent quality: a systematic comparison

For the two proteins myoglobin (MB) and fluoroacetate dehalogenase (FAcD), we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of crystals, the same sample delivery device, as well as the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins reasonable data statistics can be obtained with approximately 5000 room temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that diffraction quality is rather linked to the properties of the crystals than to the radiation source. Time-resolved experiments can therefore be conducted at the source that best matches the desired time-resolution.

biophysics↗

bioRxiv2019

Serial electron diffraction for high-throughput protein nano-crystallography

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 [A] and 1.80 [A], respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

molecular biology↗