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Review
. 2015 Mar;22(2):239-48.
doi: 10.1107/S1600577514028203. Epub 2015 Jan 29.

XFEL Diffraction: Developing Processing Methods to Optimize Data Quality

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Free PMC article
Review

XFEL Diffraction: Developing Processing Methods to Optimize Data Quality

Nicholas K Sauter. J Synchrotron Radiat. .
Free PMC article

Abstract

Serial crystallography, using either femtosecond X-ray pulses from free-electron laser sources or short synchrotron-radiation exposures, has the potential to reveal metalloprotein structural details while minimizing damage processes. However, deriving a self-consistent set of Bragg intensities from numerous still-crystal exposures remains a difficult problem, with optimal protocols likely to be quite different from those well established for rotation photography. Here several data processing issues unique to serial crystallography are examined. It is found that the limiting resolution differs for each shot, an effect that is likely to be due to both the sample heterogeneity and pulse-to-pulse variation in experimental conditions. Shots with lower resolution limits produce lower-quality models for predicting Bragg spot positions during the integration step. Also, still shots by their nature record only partial measurements of the Bragg intensity. An approximate model that corrects to the full-spot equivalent (with the simplifying assumption that the X-rays are monochromatic) brings the distribution of intensities closer to that expected from an ideal crystal, and improves the sharpness of anomalous difference Fourier peaks indicating metal positions.

Keywords: X-ray free-electron laser; mosaicity; partiality; postrefinement; serial femtosecond crystallography.

Figures

Figure 1
Figure 1
Geometric definition of partiality, accounting for the mosaic structure of the crystal. For a still shot taken with monochromatic X-rays of wavelength λ, a reciprocal lattice point (blue ball centered on Q) partially intersects the Ewald sphere. The intersection area, which is actually a spherical cap, is approximated by a circle of radius formula image, which is determined by formula image, the distance from Q to the Ewald sphere, and formula image, the resolution-dependent radius of the reciprocal lattice point as described in the text. Partiality is defined as the intersection area-to-ball volume ratio for lattice point Q, normalized by the intersection area-to-ball volume ratio of the F 000 spot at reciprocal space origin O.
Figure 2
Figure 2
Partiality estimates for Bragg spots integrated on a single thermolysin image, plotted as a function of the formula image ratio.
Figure 3
Figure 3
Resolution limits and positional accuracy of the thermolysin integration model. (a) Limiting resolution for 1000 randomly selected shots from runs 21–27 of the L498 experiment, collected at a sample-to-detector distance of 171.0 mm, and thus restricted to 2.6 Å at the detector edge, and 2.05 Å in the detector corners. Data for the strongest-diffracting samples are therefore limited by a sharp cutoff due to detector geometry rather than the intrinsic sample diffraction. Horizontal axis: limits based on bright spots picked by a spotfinding algorithm (Zhang et al., 2006 ▶); blue bars represent a histogram of resolution limits determined with ‘method 2’ from that paper. Vertical axis: limits based on a Wilson plot of the integrated intensities. (b) Displacement (in pixels) between Bragg spot positions predicted by the lattice model used for integration, and the center of mass positions actually measured for bright spotfinder-picked spots. Blue traces: displacement for 20 randomly selected shots, with bright spots from each shot grouped into resolution bins; black dots identify the highest-resolution bin for each individual shot. Red curve: aggregate displacement over the 1000 images analyzed in panel (a).
Figure 4
Figure 4
Bragg spot predictions are more accurate when the orientational model is refined against Ewald sphere distance. Two protocols are evaluated: (a) refinement of indexed spots against observed positions only, and (b) also refining the model against the angular deviation of the reciprocal lattice point from the Ewald sphere, corresponding to protocols 4 and 6 of Sauter et al. (2014 ▶), respectively. Plots represent a random sampling of processing results for simulated PSI data, in which the modeled orientation can be compared against the known true orientation from the simulation. Horizontal axis: residual misorientation angle R xy after removal of the small misorientation R z along the axis parallel to the beam direction (r.m.s. Rz misorientation is 0.017° for both panels). Vertical axis: fraction of Bragg spots predicted by the model but not present in the simulated data (blue), and fraction of Bragg spots in the simulation that are not modeled (red).
Figure 5
Figure 5
Data quality statistics for the merged structure factor intensities from thermolysin. (a, b, c) Cumulative distribution function N(L) of the local statistic: L = formula image where formula image and formula image are unrelated intensities (Padilla & Yeates, 2003 ▶). (d, e, f) Cumulative distribution function N(z), where z = I/〈I〉. Identical data were processed with the protocols listed in Table 1 ▶: (a, d) protocol 4, lattice model is not restrained against proximity to the Ewald sphere; (b, e) protocols 6 and 6F, proximity restraints are applied, with and without a separate resolution cutoff for each lattice; and (c, f) protocols 7POST and 7F,POST, which are the same as protocols 6 and 6F except that crystal orientation is postrefined to maximize agreement with a set of reference intensities as described in the text. Agreement between the merged intensities (thick lines) and the theoretical distribution (thin lines) demonstrates that such statistics offer useful metrics for evaluating different processing protocols, with the postrefined model giving the best agreement with theoretical expectation.

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