Biology subjects

Srajer, V.

Publications and source records attributed to Srajer, V..

4 recordsLinked to original sources

bioRxiv2024

Scaling and Merging Time-Resolved Laue Data with Variational Inference

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

biophysics↗

bioRxiv2024

Laue-DIALS: open-source software for polychromatic X-ray diffraction data

Most X-ray sources are inherently polychromatic. Polychromatic ("pink") X-rays provide an efficient way to conduct diffraction experiments as many more photons can be used and large regions of reciprocal space can be probed without sample rotation during exposure--ideal conditions for time-resolved applications. Analysis of such data is complicated, however, causing most X-ray facilities to discard >99% of X-ray photons to obtain monochromatic data. Key challenges in analyzing polychromatic diffraction data include lattice searching, indexing and wavelength assignment, correction of measured intensities for wavelength-dependent effects, and deconvolution of harmonics. We recently described an algorithm, Careless, that can perform harmonic deconvolution and correct measured intensities for variation in wavelength when presented with integrated diffraction intensities and assigned wavelengths. Here, we present Laue-DIALS, an open-source software pipeline that indexes and integrates polychromatic diffraction data. Laue-DIALS is based on the dxtbx toolbox, which supports the DIALS software commonly used to process monochromatic data. As such, Laue-DIALS provides many of the same advantages: an open-source, modular, and extensible architecture, providing a robust basis for future development. We present benchmark results showing that Laue-DIALS, together with Careless, provides a suitable approach to the analysis of polychromatic diffraction data, including for time-resolved applications.

molecular biology↗

bioRxiv2024

Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron

DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.

biochemistry↗

bioRxiv2020

High-resolution Crystal Structures of Transient Intermediates in the Phytochrome Photocycle

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting (dark) Pr state and the light activated Pfr state, in which light signals are received and transduced as structural change through the entire protein to modulate the activity of the protein. It is unknown how the Pr-to-Pfr interconversion occurs as the structure of intermediates remain notoriously elusive. Here, we present short-lived crystal structures of the classical phytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray Free Electron Laser 5 ns and 33ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway. The snapshots show with atomic precision how the signal progresses from the chromophore towards the output domains, explaining how plants, bacteria and fungi sense red light.

biophysics↗