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

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.

Serial femtosecond crystallography: A revolution in structural biology

“…Crystallization in vivo using insect and mammalian cells is a highly innovative approach towards nanocrystallography that has been recently discovered 6,15,59,70 . This technique was first applied in SFX in 2012 after nanocrystals were identified by electron microscopy inside insect cells when cathepsin B from Trypanosoma brucei was overexpressed using the recombinant baculovirus system 59 .…”

The birth of a new field

“…With the first 'new biology' results recently published (see papers of Liu et al [14], Gallat et al [23]), and the first highresolution time-resolved solution-scattering results from proteins about to appear (Neutze [2]), it appears that the confidence of the founders of this new field of structural and dynamic biology was not misplaced, and the US Department of Energy's gamble in building the first XFEL will pay off scientifically in many fields. New XFELs are now under construction in several countries.…”

“…It was immediately realized that the high spatial coherence provided new approaches to the phase problem, for both SPs (Loh [7], Ourmazd and co-workers [8], Martin [9], Schwander et al [16]) and nanocrystals (Millane & Chen [17], Kirian et al [18], Spence et al [19] and Barty et al [20]). Atomic resolution has so far been obtained from unknown structures only using nanocrystals (and near-atomic resolution in the FSS when small changes in a known structure are studied), so that a crucial area for development of the BioXFEL field is the development of new methods for growing nanocrystals (Kupitz et al [21], Caffrey et al [22], Gallat et al [23] and Stevenson et al [24]). When the study of the evolving damage processes (reviewed by Chapman et al [3]), diffraction physics (White [25]), simultaneous emission spectroscopy (Kern et al [15]) and detector development (Denes [26]) is added to this list of subfields, it will be seen that structure and dynamics in biology with XFELs is an extremely rich interdisciplinary field, now undergoing rapid innovation and creative development.…”

Serial femtosecond crystallography opens new avenues for Structural Biology

“…Love and P. Fromme, unpublished). Insects seem to be especially suited for in vivo crystal growth as reports also include crystal growth in the gut of living cockroaches [80,81]. Recently the first SFX structure of crystals grown inside a bacterium has been reported [38], where data were collected on isolated crystals and on crystals inside the living cells.…”

“…The data evaluation method development is constantly being improved and new features and options (like reconstruction of the peak profiles) are added and made available to all users in form of updates. The large majority of SFX structures published to date have been evaluated using Cheetah and CrystFEL [12,15,21,23,27,29,31,33,35, 36,38,61,64,67,74,76,79,81,95–98]. A second, alternate program has been developed and applied for SFX data evaluation (cctbx.xfel) [99].…”