close
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 1;73(Pt 2):93-101.
doi: 10.1107/S2053273316018696. Epub 2017 Jan 30.

Asymmetry in serial femtosecond crystallography data

Amit Sharma  1 , Linda Johansson  1 , Elin Dunevall  1 , Weixiao Y Wahlgren  1 , Richard Neutze  1 , Gergely Katona  1
Affiliations

Asymmetry in serial femtosecond crystallography data

Amit Sharma et al. Acta Crystallogr A Found Adv. .

Abstract

Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.

Keywords: Bragg reflections; ex-Gaussian distribution; intensity distribution; serial femtosecond crystallography; systematic absences.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(a)–(d) Histograms for the distribution of some of the unique Bragg reflections (yellow) and the systematically absent reflections (magenta) selected from the resolution shells around 27.3, 7.6, 4.2 and 3.8 Å, respectively, from the RCvir_XFEL data. The fits to these distributions using a Gaussian (red) and an ex-Gaussian (blue) function are shown. The fits to the Bragg reflections and the systematically absent reflections are shown as full and dashed lines, respectively. Miller indices for the reflections used are given in parentheses.
Figure 2
Figure 2
Percentage of reflections from (a) RCvir_XFEL and (b) RCvir_Sync data that could be best explained using a Gaussian (red) and an ex-Gaussian (blue) distribution.
Figure 3
Figure 3
This shows the variation in the mean (μexg, red) of the Gaussian part of an ex-Gaussian fit, the standard deviation (σexg, black), the skewness mean (τexg, blue) from the exponential part of the ex-Gaussian fit and the mean intensity of the reflection (μg, green) with the resolution for (a) RCvir_XFEL and (b) RCvir_Sync data.
Figure 4
Figure 4
Diffraction quality of RCvir_XFEL and RCvir_Sync. A representative diffraction image collected from (a) microcrystals of RCvir at the CXI (coherent X-ray imaging) beamline of the LCLS and (b) a crystal of RCvir on the ID23-2 beamline of the ESRF.
Figure 5
Figure 5
Ex-Gaussian distribution profiles using the Markov chain Monte Carlo a posteriori estimates (plots in black) for a Bragg reflection whose intensity histogram is shown in blue. Shown in red is the spread of the intensity value (I ideal) at which the c.d.f. of ex-Gaussian distributions reaches 0.95.
Figure 6
Figure 6
Wilson plots for the RCvir_XFEL data generated using Ideal Crystal (b = 1), Ideal Crystal (b = 1.05), Ideal Crystal (b = 1.19), CrystFEL _push-res = 0 process_hkl and CrystFEL partialator _model = scgaussian options are shown in grey, blue, cyan, magenta and red, respectively. Wilson plot for the RCvir_Sync data is shown in green.
Figure 7
Figure 7
Plots showing I/σ versus resolution (Å) for the RCvir_XFEL data indexed using _push-res = 0 option of CrystFEL. Plots in grey, red and magenta correspond to three different data processing methods – Ideal Crystal, CrystFEL partialator _model = scgaussian and CrystFEL process_hkl, respectively.
Figure 8
Figure 8
Figure of merit (a measure of phase quality) versus resolution plots for the RCvir_XFEL data generated using Ideal Crystal (b = 1.05), CrystFEL process_hkl, Ideal Crystal (b = 1.19) and CrystFEL partialator _model = scgaussian options are shown in blue, magenta, cyan and red, respectively.
Figure 9
Figure 9
Simulated annealing composite omit maps calculated using RCvir_XFEL data sets processed using the Ideal Crystal (b = 1.05) approach (blue) and CrystFEL process_hkl option (magenta). Selected regions of RCvir subunits are shown in four different panels: (a) residues 57–67 in cytochrome C subunit, (b) residues 47–50 in the intra-membrane subunit L, (c) residues 65–68 in the intra-membrane subunit L and (d) the mena­quinone in subunit M. Residues are shown in element colour.
See this image and copyright information in PMC

References

    1. Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221.
    1. Barends, T. R. M., Foucar, L., Botha, S., Doak, R. B., Shoeman, R. L., Nass, K., Koglin, J. E., Williams, G. J., Boutet, S., Messerschmidt, M. & Schlichting, I. (2014). Nature (London), 505, 244–247. - PubMed
    1. Boutet, S. et al. (2012). Science, 337, 362–364.
    1. Chapman, H. N. et al. (2006). Nat. Phys. 2, 839–843.
    1. Chapman, H. N. et al. (2011). Nature (London), 470, 73–77.

Substances

LinkOut - more resources