Abstract
Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the R(split) value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.
Highlights
A number of structures have been solved using serial femtosecond crystallography (SFX) at an X-ray free-electron laser (XFEL; Redecke et al, 2013; Liu et al, 2013; Boutet et al, 2012; Tenboer et al, 2014; Kern et al, 2013, 2014; Ginn et al, 2015), which have benefited from a large number of indexable snapshots from the crystalline samples
We argue that the value of the modelled partiality is acutely dependent on accurate definition of the orientation matrices to describe XFEL data, which have been gradually improving as processing techniques progress
The choice of an appropriately shaped integration box markedly improved the high-resolution information for this set of images when analysed using the methodology described in Ginn et al (2015), but errors remained at high resolution, most likely owing to residual errors in the detector geometry and the comparative weakness of the data, as well as subtle aberrations enhanced at high resolution caused by errors in detector distance and wavelength
Summary
A number of structures have been solved using serial femtosecond crystallography (SFX) at an X-ray free-electron laser (XFEL; Redecke et al, 2013; Liu et al, 2013; Boutet et al, 2012; Tenboer et al, 2014; Kern et al, 2013, 2014; Ginn et al, 2015), which have benefited from a large number of indexable snapshots from the crystalline samples. The recent structure of photoactive yellow protein (Tenboer et al, 2014), capturing time-resolved high-resolution intermediates, used between 22 678 images and 64 496 images to generate structures, achieving a discrepancy of 6.5% between two half data sets (Rsplit) for the latter. Such studies benefit from an abundant supply of crystalline sample, allowing the use of Monte Carlo integration (Kirian et al, 2010, 2011). D71, 1400–1410 research papers peaks associated with a methionine S atom were observed to a maximum of 4.5 at 3.5 Aresolution using the CrystFEL software suite (White et al, 2012; Boutet et al, 2012)
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