Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins

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.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures

“…Since then, specific damage to disulfide bonds at RT has been observed in a traditional oscillation study performed on a rotating anode X-ray generator (26), in a serial synchrotron crystallography (SSX) experiment (30) and in two multicrystal investigations (31,32). Also classifying as specific damage, the iron-water bond in a metalloprotein has been shown to elongate as a consequence of RT radiation damage at a dose as low as 0.033 MGy (33). In contrast, another multicrystal RT study on insulin did not detect any specific damage to disulfide bonds (maximum dose was 0.5 MGy) (34).…”

“…As an in-between the injection and fixed-target approaches, tape drives carrying microcrystals are yet another way of delivering crystalline samples to the X-ray beam (49,50). Together with the availability of fast readout detectors and of X-ray microbeams with increasing flux densities, these various SSX methods provide the potential to systematically study and compare both global and specific radiation damage at RT and at 100 K. Common to all of these SSX approaches is that the per-crystal X-ray radiation damage is reduced, but not eliminated, by distributing the absorbed energy per dataset over a multitude of crystals (33), as opposed to being accumulated in oscillation-based approaches.…”

Serial protein crystallography in an electron microscope

“…Minimal-damage acquisition is ensured using a dose-fractionated diffraction-duringdestruction scheme and a-posteriori critical dose determination. We have demonstrated a net acquisition rate of 35 Hz when factoring in the hit fraction, which is comparable to current liquid-jet XFEL 8,9 and synchrotron fixed-target 11,12,18 experiments. Note that a further increase of more than an order of magnitude can be achieved if dose-fractionation is omitted and acquisition speed becomes detector frame rate limited.…”

“…Sufficient signal-to-noise and completeness is achieved through merging of many thousands of such snapshots. Ideally, radiation damage effects are entirely evaded either by a "diffract-before-destroy" mode using femtosecond XFEL pulses 5 or by imposing doses too low to cause significant structural damage of each crystal, which has also been implemented at synchrotron micro-focus beam lines 11,12,18 . However, the scarcity of XFEL beamtime limits the use of protein nanocrystals for routine structure determination.…”

Serial protein crystallography in an electron microscope

“…Sufficient signal-to-noise ratio and completeness is achieved through merging of many thousands of such snapshots. Ideally, radiationdamage effects are entirely evaded either by a "diffract-beforedestroy" mode using femtosecond XFEL pulses 5 or by imposing doses too low to cause significant structural damage of each crystal, which has also been implemented at synchrotron microfocus beam lines 11,12,18 . However, the scarcity and costliness of XFEL beamtime limits the use of protein nanocrystals for routine structure determination.…”