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abstracts
The size and quality of protein crystals are critical limiting factors in conventional X-ray crystallography structural studies. Serial femtosecond X-ray crystallography (SFX) presents new opportunities through the use of submicron and nano-scale crystal samples. SFX experiments with X-ray free-electron lasers have demonstrated success (Chapman, 2011). While new instances are arising for the study of poorly crystallizing proteins, fresh examination must also be made of their analysis. Significantly, the smallness of the crystal samples creates broad integrated intensity distributions that require careful consideration for the accurate reconstruction of high-resolution protein crystal structures. Structural imperfections are also expected to play significant roles in the formation of SFX diffraction patterns (Dilanian, 2013). We present preliminary results from a new approach for SFX data analysis. As motivation, it is observed that significant similarities lie between merged SFX data-sets and powder diffraction patterns. The merged Bragg peak-shape distributions in SFX diffraction patterns are characterised by the shape, size and disorder distributions of the large crystal sample set. Powder diffraction peak-shape distributions are formed similarly by a collection of independent scatterers of such varying characteristics. The presented method utilizes an established powder diffraction analysis tool, the Le Bail method (Le Bail, 1988), for extension and application to SFX data. It is shown here that the continuous whole-pattern fitting technique, combined with variable peak-shape functions, can be used to closely model simulated SFX patterns and to extract integrated intensities. The presented approach is posed as a new analysis method for SFX experiments in which both crystal size and structural disorder variations can be incorporated.
abstracts
The complex formed between the antimalarial drug chloroquine (CQ) and its target iron(III)protoporphyrin IX (ferriheme) in aqueous solution is of considerable importance in understanding its mechanism of antimalarial activity. Recently, convincing evidence showed that CQ induces the μ-oxo dimeric form of ferriheme in aqueous solution, but the structure of this complex in solution is uncertain.[1] Molecular dynamics (MD) simulations in aqueous solution were used to model the structure of μ-oxo ferriheme and two possible conformations of the CQ-ferriheme complex, one in which CQ π-stacked with the unligated face of ferriheme and the other in which CQ was docked between the porphyrin rings. The EXAFS spectrum of μ-oxo ferriheme obtained from frozen solution strongly supported the hydrated structure determined from MD simulation where an excellent fit to the spectrum was only obtained when incorporating waters of hydration at regions identified by computation. On the other hand, the EXAFS spectrum recorded on the dried solid of μ-oxo ferriheme required no solvating waters to produce excellent agreement with the crystal structure of μ-oxo ferrihaem dimethyl ester.[2] The EXAFS spectrum recorded on a frozen aqueous solution of the CQ-ferriheme complex was able to distinguish between the two conformations modeled computationally. Fits to the EXAFS spectrum using the π-stacked structure produced poor agreement while those obtained using the docked conformation reproduced the spectrum well. This indicated that the latter is the most likely form of the CQ-ferriheme complex in aqueous solution.
