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. 2014 Jul 17;369(1647):20130497.
doi: 10.1098/rstb.2013.0497.

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

François-Xavier Gallat  1 , Naohiro Matsugaki  1 , Nathan P Coussens  2 , Koichiro J Yagi  3 , Marion Boudes  4 , Tetsuya Higashi  5 , Daisuke Tsuji  5 , Yutaka Tatano  6 , Mamoru Suzuki  7 , Eiichi Mizohata  8 , Kensuke Tono  9 , Yasumasa Joti  9 , Takashi Kameshima  9 , Jaehyun Park  10 , Changyong Song  10 , Takaki Hatsui  10 , Makina Yabashi  10 , Eriko Nango  10 , Kohji Itoh  5 , Fasséli Coulibaly  4 , Stephen Tobe  3 , S Ramaswamy  11 , Barbara Stay  12 , So Iwata  13 , Leonard M G Chavas  14
Affiliations

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

François-Xavier Gallat et al. Philos Trans R Soc Lond B Biol Sci. .

Abstract

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

Keywords: X-ray free-electron laser; in vivo crystallography; serial femtosecond crystallography.

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Figures

Figure 1.
Figure 1.
(a) Evolution of the PDB in terms of the total number of coordinates deposited each year (dark-red bar chart) and the total number of coordinates originating from data collected at synchrotron X-ray sources (blue line graph). The inset represents the total number of coordinates originating from data collected at XFELs (green bar chart, right) with the corresponding number of coordinates deposited each year (red bar chart, left). (b) Molecular weight distribution of the coordinates in the PDB (red bar chart). Within the insets are presented the structures of each molecule for which XFEL data were used (PDB accession numbers 4ac5 [2], 3pcq [3], 4fby [4] and 4hwy [5]). (Online version in colour.)
Figure 2.
Figure 2.
In vivo grown crystals enclosed inside mammalian CHO cells (a) and after extraction from human HEK293FT cells (inlet). The crystals vary in size and can reach dimensions of 15 × 15 × 3 μm3. White (top right) and yellow (bottom left) arrows point towards square- and needle-like crystals, respectively. The scale bar on the inlet picture represents 10 μm. (Online version in colour.)
Figure 3.
Figure 3.
Typical diffraction picture for the in vivo crystals of the mammalian neuraminidase hNeu1 (a) and the cockroach milk protein (b). hNeu1 crystals diffracted to 3.0 Å resolution, and cockroach milk protein crystals to 1.6 Å. The contrast of the picture was adapted to optimize visualization of the diffraction spots. (Online version in colour.)
See this image and copyright information in PMC

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