Biology subjects

Mehrabi, P.

Publications and source records attributed to Mehrabi, P..

8 recordsLinked to original sources

bioRxiv2026

Best practices for cryo-trapping time-resolvedcrystallography with the Spitrobot crystal plunger

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

biochemistry↗

bioRxiv2025

SPITROBOT-2 advances time-resolved cryo-trapping crystallography to under 25 ms

We previously introduced the SPITROBOT, a protein crystal plunging system that enables reaction quenching via cryo-trapping with a time resolution in the millisecond range. Here we present the next generation, SPITROBOT-2, as an integrated benchtop device, condensed to approximately an A4 footprint. The user experience has been enhanced by the integration of a guiding beam and a sample-switch dial, to optimise sample exchange operations. This is complemented by a light-indicated liquid nitrogen level sensor, ensuring enhanced reliability. Moreover, a fully automated shutter shields the liquid nitrogen from the humidified environment, improving sample integrity. These improvements reduce the net sample preparation time to approximately three minutes per sample. Most importantly, the cryo-trapping delay time has been reduced to 23 milliseconds, making SPITROBOT-2 twice as fast as the previous generation. This further expands the number of target systems that can be addressed by cryo-trapping time-resolved crystallography. We demonstrate successful cryo-trapping via ligand binding and conformational changes using 12 crystal structures of three independent model systems: xylose isomerase, human insulin and bacteriophage T4 lysozyme. Taken together, these improvements increase the convenient access to cryo-trapping, time-resolved X-ray crystallography empowering the MX community with efficient tools to advance research in structural biology.

biochemistry↗

bioRxiv2025

A mutation at the dimer interface regulates catalysis

Fluoroacetate Dehalogenase (FAcD), a homodimeric dehalogenase of interest in bioremediation, displays half-of-the-sites reactivity, requiring allosteric communication between the subunits to coordinate the reaction. Dimer interfaces are an obvious site of interest for understanding communication between subunits and have been shown, in a variety of enzyme systems, to mediate such communication. However, mutations at these interfaces often need to be substantial to noticeably affect protein activity. In this study, we demonstrate that two subtle interface mutations of FAcD, either accelerate the reaction or results in near-complete loss of activity with structural evidence of disruption at the dimer interface. By examining these variants using ultrahigh-resolution crystallography and kinetics studies, the influence of dimer interface variations in hydrogen-bonding networks on enzyme activity can be elucidated.

biochemistry↗

bioRxiv2022

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with millisecond time-resolution. Canonical micromesh loops are mounted on an electropneumatic piston, reactions are initiated via the liquid application method (LAMA), and finally intermediate states are cryo-trapped in liquid nitrogen. We demonstrate binding of several ligands in microcrystals of three enzymes, and trapping of reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase.

biophysics↗

bioRxiv2021

An environmental control box for serial crystallography enables multi-dimensional experiments

We present an environmental enclosure for fixed-target serial crystallography, enabling X-ray diffraction experiments in a temperature window from below 10 {degrees}C to above 70 {degrees}C - a universal parameter of protein function. Via 5D-SSX time-resolved experiments can now be carried out at physiological temperatures, providing fundamentally new insights into protein function. We show temperature-dependent modulation of turnover kinetics for the mesophilic {beta}-lactamase CTX-M-14 and for the hyperthermophilic enzyme xylose isomerase.

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↗