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In order to prolong the life-time of crystals in an X-ray beam, modern macromolecular crystallography is mainly performed using cryogenically cooled samples. However, in many cases flash cooling can degrade crystal quality if cryoprotection conditions are not ideal. Annealing or tempering of samples adversely affected by cryocooling can often, at least partially, restore diffraction quality and is now considered a standard technique in macromolecular crystallography. A small, inexpensive, automatically operated annealing device that is installed on all ESRF macromolecular crystallography beamlines is presented here. The device allows,viathe beamline control graphical user interface mxCuBE, the flash annealing of samples either from the beamline control cabin or by users accessing the beamline remotely.

X-ray fluorescence (XRF) measurements on solutions or crystals of biological macromolecules provide additional information that can be used in structure determination and/or refinement protocols. Here details are presented of an experimental setup, employed on all the ESRF Macromolecular Crystallography Group beamlines, that allows the online collection and qualitative analysis of XRF spectra. This experimental setup uses a highly attenuated beam and short exposures, meaning it is minimally destructive but retains high sensitivity.

New software for the integration of weak and/or spatially overlapped diffraction patterns is presented. The program, named PROW (profile fitting for overlapped and weak data), uses box integration with an optimized summation area for strong nonoverlapped spots, and profile fitting with an optimized fitting area for weak and/or overlapped spots. The sizes of the integration areas are dynamically adjusted to find the best compromise between minimal bias resulting from statistical fluctuations of the X-ray background and maximal use of the information content in each diffraction spot. Deconvolution of spatially overlapped spots is performed by a standard least-squares procedure. The program is compatible with the Daresbury Laue software suite and with the DENZO package. It has been tested on several data-sets recorded on image-plate and CCD (charge-coupled device) detectors. Intensities extracted from very weak Laue data-sets containing as many as 60% overlapped spots were shown to be of sufficient quality to lead to an accurate structure refinement. Difficult monochromatic data-sets were also processed successfully, leading to a generally better compromise between reliability factors, redundancy and completeness than previously achieved. Concomitant improvement in atomic model refinement was also observed.