Cryogenic coherent X-ray diffraction imaging of biological particles at SACLA

Coherent X-ray diffraction imaging (CXDI) is a promising technique to visualize internal structures of whole biological cells without sectioning. Utilizing X-ray free-electron laser (XFEL) for CXDI has potential to collect huge number of projected electron densities of such samples at higher resolutions than that limited by radiation damage. For biological application of XFEL-CXDI, sample particles must be kept in a hydrated state to maintain their functional structures, but be placed in vacuum to obtain weak diffraction signals. In addition, we need to deliver fresh sample particles one after another for XFEL exposure because every particle explodes just after X-ray irradiation. To handle these problems, we have been developing a system to fulfill cryogenic XFEL-CXDI of frozen-hydrated specimens. For cryogenic XFEL-CXDI, we prepare frozen-hydrated specimens by plunge-freezing sample particles dispersed onto thin film with humidity-controlling [1]. The diffraction experiments are conducted by using the cryogenic X-ray diffractometer KOTOBUKI-1 [2]. In a vacuum chamber of KOTOBUKI-1, a cryogenic pot equipped on a goniometer is filled with liquid nitrogen, which cools the specimen via thermal contact. Thus, KOTOBUKI-1 allows data collection at a specimen temperature of ~66 K with a positional fluctuation of less than 0.4 μm. A small angle-resolution of better than 500 nm is attainable by using a pair of L-shaped Si-slits placed before the specimen, which eliminate almost all parasite scattering from upstream. Diffraction patterns recorded on two MPCCD detectors in tandem arrangement are automatically processed and phase-retrieved by program suite SITENNO [3]. In our recent experiments performed in Japanese XFEL facility SACLA, we were able to collect a large number of diffraction patterns from biological samples at a resolution of 50 - 30 nm at an XFEL hit-rate of 20 - 100%. We report details of the cryogenic XFEL-CXDI and introduce imaging of chloroplasts as an example.

Agarose Hydrogel Protects Protein Crystals From Osmotic Shock

High-throughput protein X-ray crystallography offers an unprecedented opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional (3D) structure of the protein-ligand complex by soaking the ligand in apo-crystals, but many lead compounds are not readily water-soluble. Such lead compounds must be dissolved in concentrated organic solvents such as DMSO. Therefore, to date, it has been impossible to produce crystals of protein-ligand complexes by soaking in apo-crystals, because protein crystals dissolve immediately upon soaking in concentrated organic solvents containing lead compounds. The problem arises from the influence of osmotic shock on crystal packing during soaking. We propose an approach to avoid the damage by growing protein crystals in a high-strength hydrogel(1-3). Interestingly, the hydrogel-grown crystals did not dissolve at all for more than thirty minutes in concentrated organic solvents and ionic-strength solutions such as 60% DMSO, and 5.0M lithium acetate. Their X-ray diffraction data were suitable for structure analysis. Surprisingly, some of the crystals diffracted to the highest resolution reported in the Protein Data Bank. Furthermore, the 3D structure determined from hydrogel-grown apo-avidin crystals which were transferred to a solution containing the ligand revealed a clear electron-density map of the ligand bound to the active site. This result indicates that it is possible to bind ligand compounds into hydrogel-grown apo-crystals.

Structural analyses of the two cysteine desulfurases from Aquifex aeolicus

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer to a wide range of acceptor proteins. IscS degrades L-cysteine into L-alanine and a sulfur atom in a pyridoxal 5'-phosphate (PLP) dependent manner. In this reaction, it is essential for a conserved Lys residue of IscS to form Schiff base (the covalent bonding interaction) with PLP. Recent accumulations of genomic information have revealed that some IscS homologues in archaea and thermophilic bacteria lack this invariant Lys. Here we report the crystal structures of two paralogous cysteine desulfurases, the canonical Aa IscS1 and the invariant Lys lacking Aa IscS2, from Aquifex aeolicus. Aa IscS1/Aa IscS2 were overproduced in E. coli, and purified by heat-treatment and several column chromatography, and crystallized. The structure of Aa IscS1 was determined at 2.00 Å (Rcryst= 19.4% and Rfree = 22.0%), and Aa IscS2 at 2.55 Å (Rcryst= 21.8% and Rfree = 27.0%). Overall structures as well as orientations of the residues in the active site were quite similar to each other. In Aa IscS1 the PLP adduct was anchored in the catalytic pocket of Aa IscS1 by the formation of the aldimine Schiff base with the invariant Lys. Whereas in Aa IscS2 the PLP was not seen in the active pocket, since the catalytic Lys was substituted by Leu. Alternatively, an electron density derived from unknown-small molecule was located in the catalytic site of Aa IscS2. The shape of this electron density was completely different from that of PLP. The Bijvoet difference map calculated from data collected at λ=1.7 Å overlapped with the electron density observed in the active site; the unknown-small molecule probably contains such metals as iron atoms. Furthermore, the ICP-MS analysis demonstrated that as-isolated Aa IscS2 harbored the iron atom in the solution state. More recently we obtained the experimental evidences that non-canonical Aa IscS2 was able to form the binary complex with Aa IscU, which is responsible for a scaffold for the assembly of a nascent Fe-S cluster. Base on the structural/biochemcal results, possible physiological functions of two cysteine desulfrurases will be discussed.