Time-resolved Data Collection Research Articles

PH-dependent reaction triggering in PmHMGR crystals for time-resolved crystallography

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.

Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources.

Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.

Single grain infrared photoluminescence (IRPL) measurements of feldspars for dating

Existing infrared photoluminescence (IRPL) systems have used pulsed infrared stimulation (~830 nm) and measured IRPL emission (at 880 or 955 nm) using time resolved data collection with photomultipliers. Breakthrough of the infrared stimulation light overwhelms the IRPL, but the delayed emission during the laser-off period has been used instead.This paper describes a system for measurement of the IRPL signal from single sand-sized grains of feldspar. The attachment uses an electron-multiplying charge-coupled device (EMCCD) imaging system, and has two innovations that make it possible to use such a detector to obtain IRPL data. First, the optical detection system has been designed to minimise stray light and maximise the efficiency with which filters reject the stimulation light. This acts to reduce, but not eliminate, the breakthrough. Second, by placing the sample to be measured in a clearly defined sample grid, the spatial resolution provided by the EMCCD has been used to differentiate between regions of the image where IRPL is emitted and adjacent regions where only breakthrough is expected. This allows quantification of the breakthrough and effective subtraction to isolate the IRPL signal from the grains of interest.The attachment has been used to measure IRPL from single sand-sized grains of feldspar from an aeolian dune from New Zealand. A 1W UV LED (365 nm) is also added to the system and this is effective at resetting the IRPL signal, permitting a single aliquot regenerative dose (SAR) protocol to be used to measure equivalent dose (De). Measurement of a known laboratory dose (104 Gy) demonstrates the reproducibility of the attachment, with no overdispersion observed in the resulting single grain De values. The recovered dose is within 10% of the given dose. The natural IRPL signal yields De values from single grains with low overdispersion (22%) and giving a weighted mean value (103 ± 5.8 Gy) that is consistent with that obtained using post-IR IRSL measurements (105 ± 3.8 Gy). The attachment described here provides IRPL measurements on single grains suitable for exploring the potential of this novel and exciting signal for dating geological sediments.

The HARE chip for efficient time-resolved serial synchrotron crystallography.

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.

Logarithmic based optical delay for time-resolved data collection

A method has been established that generates values spaced according to a mathematical function, specifically the logarithm function that can be applied to a stepper motor. Here, it is applied to yield logarithmically spaced time delay points for subnanosecond interferometric time-resolved experiments using a stepper motor controlled translation stage. Application of this method is discussed in terms of three input parameters: the optical delay stage time resolution, dt; the time of maximum delay, d(stop); and the desired number of data points, N. The method improves the efficiency of interferometric time-resolved data collection while providing data collection effective to determine decay parameters. In principle, this technique could be generalized to any mathematical function.

Quantitative Analysis of Adsorbate Concentrations by Diffuse Reflectance FT-IR

Fully quantitative analyses of DRIFTS data are required when the surface concentrations and the specific rate constants of reaction (or desorption) of adsorbates are needed to validate microkinetic models. The relationship between the surface coverage of adsorbates and various functions derived from the signal collected by DRIFTS is discussed here. The Kubelka-Munk and pseudoabsorbance (noted here as absorbance, for the sake of brevity) transformations were considered, since those are the most commonly used functions when data collected by DRIFTS are reported. Theoretical calculations and experimental evidence based on the study of CO adsorption on Pt/SiO2 and formate species adsorbed on Pt/CeO2 showed that the absorbance (i.e., = log 1/R', with R' = relative reflectance) is the most appropriate, yet imperfect, function to give a linear representation of the adsorbate surface concentration in the examples treated here, for which the relative reflectance R' is typically > 60%. When the adsorbates lead to a strong signal absorption (e.g., R' < 60%), the Kubelka-Munk function is actually more appropriate. The absorbance allows a simple correction of baseline drifts, which often occur during time-resolved data collection over catalytic materials. Baseline corrections are markedly more complex in the case of the other mathematical transforms, including the function proposed by Matyshak and Krylov (Catal. Today 1995, 25, 1-87), which has been proposed as an appropriate representation of surface concentrations in DRIFTS spectroscopy.

Real-time parameterization of electrochemical machining by ultrasound measurement of the interelectrode gap

This paper discusses the development of an ultrasound technique that enables the continuous, uninterrupted collection of time-resolved data for dissolution valency, interelectrode gap, and overpotential during electrochemical machining (ECM). These parameters, in combination with the dissolution current, give insight into the ECM process. The accuracy and expediency of this approach has been established using results collected from the ECM of the stainless steel SS410. The enhanced accuracy and resolution offered by this approach has been demonstrated through comparison with established current-time and interelectrode gap-time analysis methods. This superiority has been explained both in terms of the relative accuracy of the analysis methods and the removal of the requirement for the approximation of constant-valency machining. The resulting current-overpotential relationships are sufficiently accurate to give insight into the ECM dissolution characteristics of the steel, as well as providing an improved parameter base essential for increasingly accurate simulation of the ECM process and tool design techniques.

Distribution analysis of the photon correlation spectroscopy of discrete numbers of dye molecules conjugated to DNA

The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.

Laue image analysis. III. The collection and analysis of deliberately streaked Laue exposures

The translation of the detector in its plane during the collection of a Laue diffraction pattern will draw out each diffraction spot into a streak, where the position along the streak can be related to the time course. Systematic and reliable methods of data collection and analysis have been developed to extend and optimize the amount of data obtained from streaked Laue exposures for use in time-resolved studies. Relative times along the streaks are readily established and the merging together of streak data from different crystals, with different streak lengths and exposure times, shows that absolute times can also be accurately established. While other methods of time-resolved data collection may be more appropriate in many cases, the development of the streak method does provide an attractive alternative, particularly for fast reactions where crystal disorder remains small throughout the time course.

X-Ray Position Detectors and Dynamic Structure Studies

The article reviews X-ray position detectors for dynamic diffraction works discussed by the author in the award talk, November 1985. After a resume of author's contributions in the development of such detectors as linear proportional counters, associated time-resolved data-collection techniques and area-detector diffractometers, the design and performances of direct- and indirect-type X-ray imaging TV detectors are discussed. Two major applications reviewed are real-time display of diffraction topographs and rapid measurement of reflection intensities from single crystals. As a recent progress the author describes a cylindrical position-sensitive streamer detector developed for powder works: this detector, covering a 2θ range of 120° with an angular resolution of 0.05°, can give high-quality powder patterns from specimens at high temperatures and is most adequate in structure refinement of inorganic compounds for electronic ceramics.

A Neutron Spectrometer with a Two-dimensional Detector for Time-resolved Studies

An intermediate resolution neutron spectrometer for collecting diffraction data with the range Q = 0�01-3�0 � has been built for analysing the structural organization of membranes, polymers and other molecular aggregates. This spectrometer has a position-sensitive detector with a capability for time-resolved data collection which may be used to study conformational changes in dynamical systems. A double multilayer monochromator assembly is used to monochromate neutrons. Operating wavelehgths of the spectrometer will be from 2 to 5 �, and the wavelength bandwidth (??/?) can be adjusted within the 1%-8% range.

Factors affecting the equatorial X-ray diffraction pattern from contracting frog skeletal muscle.

Changes in the equatorial X-ray diffraction pattern from tetanized frog sartorius muscles (Rana catesbiana ) were studied by use of time-resolved data collection technique (time resolution, 0.5 sec) to give information about the dynamic properties of the cross-bridges. No significant changes in the intensity ratio of two equatorial reflections (I1,0/I1,1) were observed when isometrically contracting muscles were slowly stretched by 5-6%, in spite of marked force changes. The intensity ratio also showed no significant changes when the load on isometrically contracting muscles was suddenly increased from Po to 1.2-1.5 Po to produce isotonic muscle lengthening. Closer examination of the data indicated that a small decrease in the value of I1,1 was caused by both slow stretch and isotonic lengthening. Because of the scatter of experimental plots in I1,0, the effect of small change in I1,1 on the intensity ratio fell within the range of accuracy of measurement. It is suggested that no marked changes in myosin head orientation or in the number of the cross-bridges in the vicinity of the thin filaments take place in response to slow stretches or isotonic lengthening, and that the decreased regularity of the filament lattice may produce the change in I1,1.

Low-temperature and time-resolved protein crystallography using synchrotron radiation

Internal motions in protein molecules may be investigated by stationary and time-resolved crystallography using synchrotron radiation and crystal cooling to low temperatures. The experimental techniques involved in such studies, and first results of applications to structure determination to high resolution at temperatures down to 100 K, and to time-resolved data collection on a submillisecond time scale for carbonmonoxy myoglobin following laser photolysis of the ligand, are described.