An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.
Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes.In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxidetoform the catalytic intermediate compound I(Fe IV = Oa nd ap orphyrin cation radical). Using serial femtosecond X-rayc rystallography (SFX), we have determined the pristine structures of the Fe III and Fe IV =Oredox states of aB-type DyP.These structures reveal aw ater-free distal heme site that, together with the presence of an asparagine,imply the use of the distal arginine as ac atalytic base.Acombination of mutagenesis and kinetic studies corroborate such ar ole.O ur SFX approach thus provides unique insight into howt he distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxideh eterolysis.
Some ideas and methods on how to produce high-quality samples for successful serial crystallography experiments are presented. The methods here described are aimed at experimenters trying to convert their vapour diffusion crystallization conditions into large-scale batch micro-crystallization.
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