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.

Case studies of sulfur SAD phasing with LigM from Sphingobium sp. SYK-6

Sphingobium sp. SYK-6 grows on a lignin-related biphenyl compound as the sole carbon and energy source and was initially isolated from a pond waste liquor from a kraft pulp mill. In SYK-6, 5-CH3-H4folate is synthesized from aromatic compounds such as a vanillate by a O-demethylase, LigM. The 5-CH3-H4folate is then converted to 5,10-CH2-H4folate, which is utilized for syntheses of DNA, repair DNA, and methylate DNA as well as to act as a cofactor in certain biological reactions, by another enzyme, MetF. In other bacterial speceis, 5,10-CH2-H4folate is directly synthesized by T- and H-proteins that are enzymes in Glycine Cleavage System. It is considered that SYK-6 has evolved to acquire this unique pathway for the 5,10-CH2-H4folate production, in order to survive in extreme environmental condition. To elucidate the molecular mechanisim of this pathway, we have carried out the structural analysis of LigM. LigM was purified by using IMPACT system provided from NEB, which use intein and affinity chitin-binding tag. After crystallization screening, a reservoir solution of 0.2 M Mg acetate, 0.1 M Acetate buffer pH 4.6, and 20 %(w/v) PEG8000 gave a needle crystals with approximate dimensions of 0.3×0.1×0.01 mm3. A diffraction data set was collected with 1.1 Å wavelength at BL1-A in the Photon Factory. However, phasing trials via molecular replacement (using a model with 19% sequence identity) failed. Because LigM is a 53 kDa protein and contains fourteen sulfur atoms, LigM is an interesting candidate for SAD phasing with sulfur (S-SAD). Diffraction data sets of LigM crystals were collected with 1.9 and 2.7 Å wavelengths, reaching a maximum resolution of 2.3Å. Preliminary results are promising for solving the phase problem via S-SAD. This study is also of methodological interest as the phasing capability of two different wavelengths can be compared. A thorough analysis of the diffraction data is in progress.

SAD experiment using the wavelength of 2.7 Å or longer

Single-wavelength anomalous dispersion (SAD) experiment with light atoms as anomalous scatterers has been carried out using longer wavelengths up to 2.3 Å. We have been developing a synchrotron beamline dedicated to the SAD experiments where wavelengths longer than 2.7 Å are available to enhance weak anomalous signals. Larger background noise due to the longer wavelength, which is one of the major problems in the experiment, is reduced by introducing a standing helium chamber surrounding both the whole diffractometer and the X-ray detector. The system allows to perform experiments with normal and long waveldngths under the same environment. Helium cold stream is fed into the chamber at the sample position and reused after removing contaminants to keep the temperature of the stream at 30 K or below economically. Capillary-top-mount method [1] was improved to further reduce the background noise and to accommodate with smaller or needle-shape crystals. Several results on de-novo structural solutions with sulfur-SAD phasing will be reported in addition to the current performance of the beamline and its future plan.