Softer but stronger? Sulfur SAD with low energy X-rays of 2.7 Å

Single wavelength anomalous diffraction (SAD) is a powerful experimental phasing technique used in macromolecular crystallography (MX). SAD is based on the absorption of X-rays by heavy atoms, which can be either incorporated into the protein (crystal) or naturally present in the structure, such as sulfur or metal ions. In particular, sulfur seems to be an attractive candidate for phasing, because most proteins contain a considerable number of S atoms. However, the K-absorption edge of sulfur is around 5.1 Å wavelength (2.4 keV), which is far from the optimal wavelength of most MX-beamlines at synchrotrons. Therefore, phasing experiments have to be performed further away from the absorption edge, which results in weaker anomalous signal. This explains why S-SAD was not commonly used for a long time, although its feasibility was illustrated by the ground-breaking study by Hendrickson and Teeter [1]. Recent developments in instrumentation, software and methodology made it possible to measure intensities more accurately, and, as a consequence, S-SAD has lately obtained more and more attention [2]. The beamline BL-1A at Photon factory (KEK, Japan) is designed to take full advantage of a long wavelength X-ray beam at around 3 Å to further enhance anomalous signals. We performed S-SAD experiments at BL-1A using two different wavelengths (1.9 Å and 2.7 Å) and compared their phasing capabilities. This methodological study was performed with ferredoxin reductase crystals of various sizes. In order to guarantee statistical validity and to exclude the influence of a particular sample, we repeated the comparison with several crystals. The novelty in the approach consists in using very long wavelengths (2.7 Å), not fully exploited in the literature so far. According to our study, the 2.7 Å wavelength shows - despite strong absorption effects of the diffracted X-rays - more successful phasing results than at 1.9 Å.

Structure and function of the CagA oncoprotein from Helicobacter pylori

CagA is known as a major bacterial virulence determinant from Helicobacter pylori and is critical for gastric cancer. Upon delivery into the gastric epithelial cells, CagA localizes to the inner leaflet of the plasma membrane and promiscuously interacts with host proteins such as PAR1b and SHP2. The CagA-PAR1-SHP2 complex potentiates oncogenic signaling. Biochemical and physicochemical analyses revealed that CagA is comprises a structured N-terminal region (residues 1-876) and an intrinsically disordered C-terminal region (residues 877-1186). To understand the structure and function relationship of CagA, we determined the crystal structure of the N-terminal region (residues 1-876) of CagA [1]. The N-terminal CagA is rich in α-helices and composed of three domains. Domain I (residues 24-221) is linked to domain II (residues 303-644) by a disordered loop with about 80 amino acid residues. Domain II has a basic patch composed of 14 lysine and 2 arginine residues. Biological experiments revealed that the basic patch mediates the CagA-phosphatidylserine interaction to localize the inner face of the plasma membrane. In addition, we found that C-terminal disordered region forms a lariat-like loop by the interaction between NBS (residues 645 - 824) and CBS (residues 998 - 1038) in the disordered C-terminal region. The formation of the lariat-like loop facilitates promiscuous interaction of CagA with target protein such as SHP2.

Anaerobic crystallization for protein crystallography

Crystallization has been a bottleneck in protein crystallography. Major problems in protein crystallization are 1) to find crystallization conditions effectively at the initial crystallization screening and 2) to improve the reproducibility of protein crystallization. To overcome these problems, we have proposed some techniques such as the immediate observation method (1). Recently, we realized that films and precipitates of oxidized proteins hampered the crystal formation, leading to poor reproducibility of the crystallization. To avoid oxidation of proteins, we examined anaerobic crystallization in an anaerobic chamber. The anaerobic chamber (Anaerobic `HARD', Hirasawa) was designed to carry out controlled anaerobic experiments for electron-transfer proteins. We have so far established typical procedures for the anaerobic crystallization (2). On the basis of our earlier experiences, the anaerobic crystallization was tested for various proteins. We found obvious differences between aerobic and anaerobic crystallization in some cases; some proteins could crystallize only under anaerobic conditions. Furthermore, the anaerobic crystallization improved reproducibility of crystallization as expected. We will report some examples of the anaerobic crystallization.