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

Abstract

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D_1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.

Keywords: X-ray radiation damage; room temperature synchrotron data collection; serial crystallography.

Fig. 1.

Serial radiation damage raster-scanning approach. Protein crystals (cf. photo) are sandwiched between two Si₃N₄ membranes and presented to the X-ray beam on a solid support (30) for RT data collection or deposited on a microporous silicon chip (52) for data collection at 100 K maintained by a stream of gaseous nitrogen. As an example, the scheme shows the RT high-dose rate series for which two Si₃N₄ sandwiches were used. The support was raster scanned across the micrometer-sized X-ray beam from the upper left to the lower right with each line being scanned from left to right. Each Si₃N₄ membrane is sampled at a total number of 140 (x) × 140 (y) positions, i.e., at 19,600 positions. At each position sampled (indicated in the photo by white circles for the first three lines), 40 consecutive still diffraction patterns are retained from the 50 collected by truncating the first 5 and last 5 as noted in the text, each for a 2.01-ms exposure time with an increasing average X-ray dose on a high-frame rate EIGER-X 4M detector. The 1st, 9th, and 28th positions are highlighted. Sampled positions are spaced by 10 μm, horizontally and vertically. The small whitish dots uniformly observed along the x and y axes of the Si₃N₄ membrane (see photo) are marks left by the X-ray beam. They provide an estimate of the extent to which visual radiation damage spreads. Patterns of equivalent dose collected at 19,818 positions (i.e., corresponding to 19,818 indexed hits, from multiple membranes) are then assembled into 40 equivalent-dose serial-crystallography datasets from which protein structures at increasing dose are solved.

Fig. 2.

Decrease in diffraction power as a function of increasing average dose. The sum of the intensities of all reflections up to the detector edges in all indexed diffraction patterns of a dataset, normalized by the sum of the first (i.e., lowest dose) dataset are shown as a function of the average dose delivered by a Gaussian beam (AD_G95, average over the region of the crystal where 95% of the energy is deposited) for dose rates of 2.4 MGy/s (circles) and 40.3 MGy/s (triangles) at RT and of 40.3 MGy/s (squares) at 100 K. The dose at which the diffracted intensity decreased to one-half of its initial value (D_1/2) was determined to be 0.36, 0.57, and 15.3 MGy for the RT series at 2.4 and 40.3 MGy/s and the cryo- series at 40.3 MGy/s, respectively.

Fig. 3.

Modeling and simulation of the decrease in diffraction power for the RT series at 40.3 MGy/s. (A) The sum of the intensities of all predicted reflections for each indexed pattern of a dataset n was averaged and normalized by the sum of the first (i.e., lowest dose) dataset (triangles, same data as in Fig. 2). The fit to these data according to a three-beam model (hot, cold, and nondamaging beams; see SI Appendix, Supplementary Text S1 for details) approximating the Voigt-shaped beam is shown as a dashed line. The dotted line is the per-voxel simulation of I_n/I₁, with the Voigt-shaped beam represented by the sum of a Gaussian and a Lorentzian profile (see SI Appendix, Supplementary Text S2 for details). (B) Relative contributions of the hot (SI Appendix, Eq. S9) and cold (SI Appendix, Eq. S10) beams of the three-beam model as a function of average dose. Only these two beams have been used in the weight computation that will further be used in the model of the specific damage (SI Appendix, Eq. S11). Indeed, as the third-beam contribution to the diffraction intensity is assumed to be constant in the dose range studied, it does not generate features in F_o^RT-HDRn – F_o^RT-LDR1 maps.

Fig. 4.

Specific radiation damage to disulfide bonds as a function of increasing average dose. Sequential difference Fourier maps at the disulfide bond Cys6–Cys127 (A) between the 43rd (3.47 MGy) and the 1st datasets in the cryo- high-dose rate series contoured at ±0.06 e⁻/ų (negative and positive peaks are in red and green, respectively), (C) between the 6th dataset (0.48 MGy) in the RT 40.3 MGy/s series and the 1st dataset in the RT 2.4 MGy/s series (at ±0.06 e⁻/ų), and (E) between the 80th (0.39 MGy) and the 1st dataset in the RT 2.4 MGy/s series (at ±0.06 e⁻/ų). Sum of the integrated density in Fourier difference maps around both sulfur atoms in the disulfide bonds as a function of average dose (B) for the cryo- 40.3 MGy/s, (D) RT 40.3 MGy/s and (F) RT 2.4 MGy/s series. Note, only every fifth data point is shown at doses above 0.24 MGy in F. In D, the dashed line indicates the fit result according to a “weighted specific damage” model (SI Appendix, Eq. S11) based on the Voigt-shaped beam being represented by a three-beam model (see SI Appendix, Supplementary Text S1 for details), and the dotted line, the per-voxel simulation of specific damage, with the Voigt profile describing the microbeam approximated by the sum of a Gaussian and a Lorentzian profile (see SI Appendix, Supplementary Text S2 for details). The Inset in F focuses on a reduced dose scale. Models of the cryo- 40.3 MGy/s series dataset 1 (Protein Data Bank [PDB] ID code 6Q8T) and of the RT 2.4 MGy/s series dataset 1 (PDB ID code 6Q88) are superimposed on the difference Fourier maps in A and in C and E, respectively.