Briony A. Yorke

Revealing low-dose radiation damage using single-crystal spectroscopy

Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed.

Time-resolved crystallography using the Hadamard transform

We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy.

X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection

During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals.

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase

Threonine 57 is identified as the key residue required for the post-translational activation of E. coli aspartate decarboxylase. The crystal structure of the site-directed mutant T57V is reported.

Pump-Probe Spectroscopy Using the Hadamard Transform.

A new method of performing pump–probe experiments is proposed and experimentally demonstrated by a proof of concept on the millisecond scale. The idea behind this method is to measure the total probe intensity arising from several time points as a group, instead of measuring each time separately. These measurements are multiplexes that are then transformed into the true signal via multiplication with a binary Hadamard S matrix. Each group of probe pulses is determined by using the pattern of a row of the Hadamard S matrix and the experiment is completed by rotating this pattern by one step for each sample excitation until the original pattern is again produced. Thus to measure n time points, n excitation events are needed and n probe patterns each taken from the n × n S matrix. The time resolution is determined by the shortest time between the probe pulses. In principle, this method could be used over all timescales, instead of the conventional pump–probe method which uses delay lines for picosecond and faster time resolution, or fast detectors and oscilloscopes on longer timescales. This new method is particularly suitable for situations where the probe intensity is weak and/or the detector is noisy. When the detector is noisy, there is in principle a signal to noise advantage over conventional pump–probe methods.

A new approach to time-resolved X-ray crystallography

Time-resolved crystallography is able to provide four-dimensional structural information about short-lived intermediate states, with near-atomic resolution. This information can be used to elucidate molecular mechanisms relevant to areas such as drug-design, chemical and biological sensors, and energy and information storage. The current state of the art time-resolved experiments can reach picosecond time-resolutions using Laue crystallography but such experiments can only be carried out at a few beamlines worldwide.We have developed a new transform time-resolved method that can be performed using a monochromatic beamline at a synchrotron and still achieve high time-resolution, vastly increasing the accessibility of such experiments. Here we present initial results demonstrating the method.