Elemental analysis of proteins by proton induced X-ray emission (microPIXE)

The identification and quantification of metals bound to proteins is a crucial problem to be solved in structural biology. This chapter will describe the technique of proton induced X-ray emission with a microfocused proton beam (microPIXE) as a tool for analysing the elemental composition of liquid and crystalline protein samples. The proton beam induces characteristic X-ray emission from all elements in the protein, which can be interpreted in terms of the metal content of the protein molecule with a relative accuracy of between 10 and 20 %. The compelling advantage of this method is that the sulphur atoms in the methionines and cysteines of the protein provide an internal calibration of the number of protein molecules present so that systematic errors are minimised and the technique is entirely internally self-consistent. This is achieved by the simultaneous measurement of the energy of backscattered protons (Rutherford backscattering), to enable the matrix composition and thickness to be determined, and so correct the PIXE data for the self-absorption of X-rays in the sample. The technical and experimental procedures of the technique will be outlined, and examples of recent measurements given which have informed a range of investigations in structural biology. The use of the technique is increasing and we are in the final stages of developing it to be a routine high-throughput method. © 2013 Springer Science+Business Media Dordrecht

Optimizing the spatial distribution of dose in X-ray macromolecular crystallography.

X-ray data collection for macromolecular crystallography can lead to highly inhomogeneous distributions of dose within the crystal volume for cases when the crystal is larger than the beam or when the beam is non-uniform (gaussian-like), particularly when crystal rotation is fully taken into account. Here the spatial distribution of dose is quantitatively modelled in order to compare the effectiveness of two dose-spreading data-collection protocols: helical scanning and translational collection. Their effectiveness in reducing the peak dose per unit diffraction is investigated via simulations for four common crystal shapes (cube, plate, long and short needles) and beams with a wide range of full width half maximum values. By inspection of the chosen metric, it is concluded that the optimum strategy is always to use as flat (top-hat) a beam as possible and to either match the beam size in both dimensions to the crystal, or to perform a helical scan with a beam which is narrow along the rotation axis and matched to the crystal size along the perpendicular axis. For crystal shapes where this is not possible, the reduction in peak dose per unit diffraction achieved through dose spreading is quantified and tabulated as a reference for experimenters

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

International audienceSubstrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the $Arabidopsis\ thaliana$ enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I$_{320}$ intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand $\beta$6 of the Pdx1 ($\beta \alpha$)$_8$-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 ${\AA}$ between substrate- and product-binding sites

Practical radiation damage-induced phasing

International audienceAlthough crystallographers typically seek to mitigate radiation damage in macromolecular crystals, in some cases, radiation damage to specific atoms can be used to determine phases de novo. This process is called radiation damage-induced phasing or "RIP." Here, we provide a general overview of the method and a practical set of data collection and processing strategies for phasing macromolecular structures using RIP