Alternative phasing method in macromolecular crystallography

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].

Chemical Crystallography at the Australian Synchrotron MX Beamlines

The Macromolecular Crystallography (MX) Beamlines at the Australian Synchrotron collect data on protein samples (PX) and chemical samples (CX). This broad range of sample types requires us to consider a number of experimental and data processing considerations. Protein samples have very large unit cells but diffract weakly, the chemical samples on the other hand have very small unit cell and diffract comparatively strongly. From an experimental point of view, this requires substantially different geometrical considerations which can be handled by changing the energy of the monochromated X-rays and detector distance. Another consideration is detector type, the detectors at the MX beamlines are from the Area Detector Systems Corporation (ADSC) and they have generally been used for PX data collections. This has lead to investigation regarding high incidence angle phosphor thickness corrections. As the detectors have mainly been used for PX work, the software for sample handling has also been developed with PX considerations rather than CX. For example, the software for space group determinations is set my default to assumes that your space group is only ever one of the 65 space groups that don't contain mirror, inversion or glide operations. Another area of interest is the way data scaling is handled. The data is often scaled with PX data for a number of reasons, with the most common scaling of data is due t the prevalence of radiation damage to the samples. By contrast the most common form of scaling for CX data is for sample anisotropy in strong absorbers. A discussion of the challenges faced for the chemical crystallography experiments at the Australian Synchrotron will be presented.

The structure of an ABC toxin particle determined by X-ray crystallography and single particle EM.

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and sub-sequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. We have determined the three-dimensional structure of the complex formed between the B and C proteins by X-ray crystallography to 2.5Å. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B–C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a striking structural architecture that we predict to be conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery. We have been able to model the complete ABC toxin complex for the by docking the B–C complex and both associated chitinase enzymes, Chi1 and Chi2, onto the single-particle electron microscopy structure of the Y. entomophaga A pentamer. The structure of the complete complex presented here reveals how the cytotoxic C proteins of ABC-type toxin complexes are processed and protected, demonstrates the function of the B protein within the complex and provides a framework for further experiments to build a complete mechanistic model of action for this class of toxins. More broadly, it also illuminates the function of the widely distributed RHS- and YD-repeat families of proteins, which had previously been unknown.