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Abstract
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.
Highlights
Enzymology and structural biology are highly dependent on the accurate three-dimensional models obtained by X-ray crystallography
A major challenge in conventional synchrotron-based X-ray crystallography, at room temperature, is the extremely rapid onset of radiation damage, i.e. changes to the structure of the protein caused by the ionizing effects of the X-ray beam
A low-dose series of synchrotron Multiple serial structures (MSS) is anchored by a damage-free SFX structure, both being determined using the same fixed-target serial sample-delivery system. We present this approach as a general method to efficiently collect both SFX and SSX data under near-identical conditions, characterize subtle site-specific changes caused by X-rays in proteins and allow direct comparison of, and extrapolation to, damage-free X-ray free-electron lasers (XFELs) structures from low-dose synchrotron models
Summary
Enzymology and structural biology are highly dependent on the accurate three-dimensional models obtained by X-ray crystallography. Such structures provide insight into function and can form a basis for understanding how proteins interact with each other or with small molecules. The structure obtained should be representative of the native state of the protein. Macromolecular crystallography is typically carried out at cryogenic temperatures (100 K) to minimize radiation-damage-induced structural perturbation (Garman & Weik, 2017; Holton, 2009). A major challenge in conventional synchrotron-based X-ray crystallography, at room temperature, is the extremely rapid onset of radiation damage, i.e. changes to the structure of the protein caused by the ionizing effects of the X-ray beam
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