Single crystal X-ray diffraction techniques applied on biological molecules have specific requirements. The significantly larger unit cell of bio-molecular crystal results in much weaker diffraction intensities compared to small molecules and high sensitivity to radiation damage, which limits total exposure per data set. Therefore, the development of X-ray diffraction technologies was marked by important advances in the areas of X-ray sources, detectors, goniometers, automation, in situ approaches, and software.
Advances in each area broadened the possibilities of the field and pushed the limits of single crystal protein structure analysis. X-ray free electron lasers enable crystal analysis with use of submicron crystals outrunning the radiation damage events. Photon counting detectors provide one of the most efficient ways of detecting diffraction intensities. While high-precision four-circle goniometers find use in in house analysis of very small crystals, the ever growing intensity of the synchrotron beams available for macromolecular crystallography led to development of ultra-fast single-axis goniometers enabling thus several-second-data-set acquisition. Automation of crystallization and crystal manipulation simplified screening of large numbers of conditions as well as single crystals. Automated techniques of in situ (or in tray) measurements opened up screening for ligand binding (or derivatives) in solvated conditions without the need for crystal vitrification. Devices controlling crystal humidity enable optimization of macromolecular single crystals as for their packing and quality of diffraction pattern. Recent software development enabled automated or semi-automated parallelized diffraction data processing providing quick answers in the case of utilization of relatively costly beam times at synchrotron beam lines.
Each of the main technological areas will be briefly discussed in the light of the current limits of use in macromolecular crystallography.
Despite the enormous technological improvements the basic understanding of diffraction experiment, its parameters, and background in single crystal analysis requires relatively good understanding of the field. Excellent technology can be used incorrectly or non-efficiently if the basic principles are ignored or the modern technologies are not properly understood. While some important parameters of macromolecular crystallography can be improved or their negative effects minimized with the use of modern equipment or procedures, other critical parameters cannot be ignored. The distinction between these two types of characteristics/parameters will be discussed to support better application of macromolecular crystallography.
Support by MEYS CR (LM2015043, CIISB) and by the ERDF fund (CZ.02.1.01/0.0/0.0/16_013/0001776) is acknowledged.
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