Biology subjects

Axford, D.

Publications and source records attributed to Axford, D..

9 recordsLinked to original sources

bioRxiv2026

Use of photocaged molecular oxygen for time-resolved serial X-ray crystallography

Photocages offer an attractive means of synchronously triggering enzyme-substrate driven reactions in biological systems in crystallo expanding the reach of light-driven catalysis. We describe the application of photocaged molecular oxygen to trigger molecular oxygen binding in crystals of myoglobin under anaerobic conditions and follow structural changes using both serial synchrotron and serial femtosecond X-ray crystallography. This is enabled through use of fixed targets under anaerobic conditions, utilising thin polymeric films with low molecular oxygen permeability and validated by serially collecting deoxy myoglobin structures and complementary in crystallo UV-Vis spectroscopy. Release of molecular oxygen from the photocage and subsequent binding of the gaseous ligand is structurally visualised in oxygen-bound structures of myoglobin at 5 and 10 ms and various laser parameters. We present a robust workflow for enabling anaerobic room-temperature data collection of oxygen-sensitive samples on fixed targets and report the successful photo-release of caged molecular oxygen for time-resolved serial crystallography.

molecular biology↗

bioRxiv2025

Integrated structural dynamics uncover new modes of B12 photoreceptor activation

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The molecular mechanism of B12 photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B12 photoreceptor CarH4,5 from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co-C bond triggers a pathway of structural changes that propagate throughout CarH from the B12 chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B12 enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B12-dependent photoreceptors for future optogenetic applications.

biophysics↗

bioRxiv2023

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin.

1Human gamma-D crystallin (HGD) is the major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin shows no post-translational modifications. After aging for nine months in the absence of light, a covalently bound reducing agent modifying surface cysteines is observed for the first time by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

biochemistry↗

bioRxiv2023

A redox switch allows binding of ferrous and ferric ions in the cyanobacterial iron binding protein FutA from Prochlorococcus

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance StatementOceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

biophysics↗

bioRxiv2022

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy*, Hill*, 2018). Here we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly -- but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryogenic-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

biophysics↗

bioRxiv2022

xia2.multiplex: a multi-crystal data analysis pipeline

In macromolecular crystallography radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals, for example small-wedge data collections on micro-crystals, in situ room-temperature data collections, and collection from membrane proteins in lipidic mesophase. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. Identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multicrystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After describing a number of use cases, we demonstrate the benefit of xia2.multiplex within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease.

molecular biology↗

bioRxiv2021

Experimental evidence for the benefits of higher X-ray energies for macromolecular crystallography

X-ray induced radiation damage is a limiting factor for the macromolecular crystallographer and data must often be merged from many crystals to yield complete datasets for structure solution of challenging samples. Increasing the X-ray energy beyond the typical 10-15 keV range promises to provide an extension of crystal lifetime via an increase in diffraction efficiency. To date however hardware limitations have negated any possible gains. Through the first use of a Cadmium Telluride Eiger2 detector and a beamline optimised for high energy data collection, we show that at higher energies fewer crystals will be required to obtain complete data, as the diffracted intensity per unit dose increases by a factor of more than 3 between 12.4 and 25 keV. Additionally, those higher energy data provide more information, evidenced by an increase in high-resolution limit of up to 0.3 [A], pointing to a high energy future for synchrotron-based macromolecular crystallography.

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

Direct measurement of X-ray induced heating of microcrystals

Temperature control is a key aspect of macromolecular crystallography, with the technique of cryocooling routinely used to mitigate X-ray induced damage. Beam induced heating could cause the temperature of crystals to rise above the glass transition temperature, greatly increasing the rate of damage. X-ray induced heating of ruby crystals 20-40 microns in size has been quantified non-invasively by monitoring the emission wavelengths of X-ray induced fluorescence during exposure to the X-ray beam. For beamsizes and dose-rates typically used in macromolecular crystallography the temperature rises are of order 20 K. The temperature changes observed are compared with models in the literature and can be used as a validation tool for future models.\n\nSynopsisX-ray induced heating of micro-crystals is quantified through the temperature-dependent shift in X-ray induced fluorescence from ruby crystals.

biophysics↗