Serial femtosecond crystallography explained

Serial femtosecond crystallography (SFX) is a form of X-ray crystallography developed for use at X-ray free-electron lasers (XFELs).^{[1] [2] [3]} Single pulses at free-electron lasers are bright enough to generate resolvable Bragg diffraction from sub-micron crystals. However, these pulses also destroy the crystals, meaning that a full data set involves collecting diffraction from many crystals. This method of data collection is referred to as serial, referencing a row of crystals streaming across the X-ray beam, one at a time.

History

While the idea of serial crystallography had been proposed earlier,^[4] it was first demonstrated with XFELs by Chapman et al.^[5] at the Linac Coherent Light Source (LCLS) in 2011. This method has since been extended to solve unknown structures, perform time-resolved experiments, and later even brought back to synchrotron X-ray sources.

Methods

In comparison to conventional crystallography, where a single (relatively large) crystal is rotated in order to collect a 3D data set, some additional methods have to be developed to measure in the serial mode. First, a method is required to efficiently stream crystals across the beam focus. The other major difference is in the data analysis pipeline. Here, each crystal is in a random, unknown orientation which must be computationally determined before the diffraction patterns from all the crystals can be merged into a set of 3D hkℓ intensities.

Sample Delivery

The first sample delivery system used for this technique was the Gas Dynamic Virtual Nozzle (GDVN) which generates a liquid jet in vacuum (accelerated by a concentric helium gas stream) containing crystals. Since then, many other methods have been successfully demonstrated at both XFELs and synchrotron sources. A summary of these methods along with their key relative features is given below:

• Gas Dynamic Virtual Nozzle (GDVN)^[6] - low background scattering, but high sample consumption. Only method available for high repetition rate sources.^[7]
• Lipidic Cubic Phase (LCP) injector^[8] - Low sample consumption, with relatively high background. Specially suited for membrane proteins
• Other viscous delivery media^{[9] [10]} - Similar to LCP, low sample consumption with high background
• Fixed target scanning systems (wide variety of systems have been used with different features, with standard crystal loops,^[11] or silicon chips^[12]) - Low sample consumption, background depends on system, mechanically complex
• Tape drive (crystals auto-pipetted onto a Kapton tape and brought to X-ray focus) - Similar to fixed target systems, except with fewer moving parts

Data Analysis

In order to recover a 3D structure from the individual diffraction patterns, they must be oriented, scaled and merged to generate a list of hkℓ intensities. These intensities can then be passed to standard crystallographic phasing and refinement programs. The first experiments only oriented the patterns^[13] and obtained accurate intensity values by averaging over a large number of crystals (> 100,000). Later versions correct for variations in individual pattern properties such as overall intensity variations and B-factor variations as well as refining the orientations to fix the "partialities" of the individual Bragg reflections.^[14]

External links

• CrystFEL
• cctbx.xfel
• NXDS
• The revolution of XFEL

Notes and References

1. Serial Femtosecond Crystallography of G Protein–Coupled Receptors - PubAg . Science . 2013 . . US . 24357322 . en . 2019-02-26. Liu . W. . Wacker . D. . Gati . C. . Han . G. W. . James . D. . Wang . D. . Nelson . G. . Weierstall . U. . Katritch . V. . Barty . A. . Zatsepin . N. A. . Li . D. . Messerschmidt . M. . Boutet . S. . Williams . G. J. . Koglin . J. E. . Seibert . M. M. . Wang . C. . Shah . S. T. . Basu . S. . Fromme . R. . Kupitz . C. . Rendek . K. N. . Grotjohann . I. . Fromme . P. . Kirian . R. A. . Beyerlein . K. R. . White . T. A. . Chapman . H. N. . Caffrey . M. . 342 . 6165 . 1521–1524 . 10.1126/science.1244142 . 3902108 . 1 .
2. Mizohata E, Nakane T, Fukuda Y, Nango E, Iwata S . Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology . Biophysical Reviews . 10 . 2 . 209–218 . April 2018 . 29196935 . 5899704 . 10.1007/s12551-017-0344-9 .
3. Martin-Garcia JM, Conrad CE, Coe J, Roy-Chowdhury S, Fromme P . Serial femtosecond crystallography: A revolution in structural biology . Archives of Biochemistry and Biophysics . 602 . 32–47 . July 2016 . 27143509 . 4909539 . 10.1016/j.abb.2016.03.036 .
4. Neutze R, etal . Potential for biomolecular imaging with femtosecond X-ray pulses . Nature . 406 . 752–757 . August 2000 . 6797 . 10.1038/35021099 . 10963603 . 4300920 .
5. Chapman HN, Fromme P, Barty A, White TA, Kirian RA, Aquila A, etal . Femtosecond X-ray protein nanocrystallography . Nature . 470 . 7332 . 73–7 . February 2011 . 21293373 . 3429598 . 10.1038/nature09750 . 2011Natur.470...73C .
6. DePonte DP, Weierstall U, Schmidt K, Warner J, Starodub D, Spence JC, Doak RB . Gas dynamic virtual nozzle for generation of microscopic droplet streams. . Journal of Physics D: Applied Physics . September 2008 . 41 . 19 . 195505 . 10.1088/0022-3727/41/19/195505 . 0803.4181 . 2008JPhD...41s5505D . 119259244 .
7. Wiedorn MO, Awel S, Morgan AJ, Ayyer K, Gevorkov Y, Fleckenstein H, etal . Rapid sample delivery for megahertz serial crystallography at X-ray FELs . IUCrJ . 5 . Pt 5 . 574–584 . September 2018 . 30224961 . 6126653 . 10.1107/S2052252518008369 .
8. Weierstall U, James D, Wang C, White TA, Wang D, Liu W, etal . Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography . Nature Communications . 5 . 3309 . 2014 . 24525480 . 4061911 . 10.1038/ncomms4309 . 2014NatCo...5.3309W .
9. Sugahara M, Mizohata E, Nango E, Suzuki M, Tanaka T, Masuda T, etal . Grease matrix as a versatile carrier of proteins for serial crystallography . Nature Methods . 12 . 1 . 61–3 . January 2015 . 25384243 . 10.1038/nmeth.3172 . 2433/203008 . 25950836 . free .
10. Conrad CE, Basu S, James D, Wang D, Schaffer A, Roy-Chowdhury S, etal . A novel inert crystal delivery medium for serial femtosecond crystallography . IUCrJ . 2 . Pt 4 . 421–30 . July 2015 . 26177184 . 4491314 . 10.1107/S2052252515009811 .
11. Gati C, Bourenkov G, Klinge M, Rehders D, Stellato F, Oberthür D, etal . Serial crystallography on in vivo grown microcrystals using synchrotron radiation . IUCrJ . 1 . Pt 2 . 87–94 . March 2014 . 25075324 . 4062088 . 10.1107/S2052252513033939 .
12. Roedig P, Ginn HM, Pakendorf T, Sutton G, Harlos K, Walter TS, etal . High-speed fixed-target serial virus crystallography . Nature Methods . 14 . 8 . 805–810 . August 2017 . 28628129 . 5588887 . 10.1038/nmeth.4335 .
13. White TA, Kirian RA, Martin AV, Aquila A, Nass K, Barty A, Chapman HN . CrystFEL: a software suite for snapshot serial crystallography. . Journal of Applied Crystallography . April 2012 . 45 . 2 . 335–41 . 10.1107/S0021889812002312 .
14. White TA, Mariani V, Brehm W, Yefanov O, Barty A, Beyerlein KR, Chervinskii F, Galli L, Gati C, Nakane T, Tolstikova A, Yamashita K, Yoon CH, Diederichs K, Chapman HN . Recent developments in CrystFEL . Journal of Applied Crystallography . 49 . Pt 2 . 680–689 . April 2016 . 27047311 . 4815879 . 10.1107/S1600576716004751 .