Marc Schiltz

Large uniaxial negative thermal expansion in pentacene due to steric hindrance

The uniaxial negative thermal expansion in pentacene crystals along a is a particularity in the series of the oligoacenes and is exceptionally large for a crystalline solid. Full x-ray structure analysis from 120 to 413 K reveals that the dominant thermal motion is a libration of the rigid molecules about their long axes, modifying the intermolecular angle which describes the herringbone packing within the layers. This herringbone angle increases with temperature by 0.3° – 0.6° per 100 K and causes an anisotropic rearrangement of the molecules within the layers, i.e., an expansion in the b direction and a distinct contraction along a. Additionally, a larger herringbone angle improves the cofacial overlap between adjacent, parallel molecules, and thus enhances the attractive van der Waals forces.

Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.

Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms

The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.

X-ray absorption, refraction and resonant scattering tensors in selenated protein crystals: implications for data collection strategies in macromolecular crystallography

Polarized fluorescence spectra were recorded in the vicinity of the Se and Br K edges on crystals of the selenated protein aldose reductase in complex with a brominated inhibitor molecule. The X-ray absorption, refraction and resonant scattering tensors as a function of X-ray energy were derived from these data. Substantial dichroism and anisotropy of resonant scattering were observed in these crystals. It is predicted that these effects are present in many resonant scattering experiments in macromolecular crystallography and are likely to affect the diffraction data. As a consequence, the anomalous phasing signal in single- or multi-wavelength anomalous diffraction experiments can be optimized simply by choosing a judicious orientation of the crystal with respect to the polarization direction of the incident X-ray beam. A simple procedure is presented to achieve this, prior to any knowledge about the selenium sites.

Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments

It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment.

Molecular envelopes derived from protein powder diffraction data

The preparation of single crystals suitable for X-ray analysis is frequently the most difficult step in structural studies of proteins.With the aid of two examples, it is shown that de novo solution of the crystallographic phase problem can be achieved at low resolution using microcrystalline powder samples via the single isomorphous replacement method. With synchrotron radiation and optimized instrumentation, high-quality powder patterns have been recorded, from which it was possible to generate phase information for structure factors up to 6 A resolution. pH- and radiation-induced anisotropic lattice changes were exploited to reduce the problem of overlapping reflections, which is a major challenge in protein powder diffraction. The resulting data were of sufficient quality to compute molecular envelopes of the protein molecule and to map out the solvent channels in the crystals. The results show that protein powder diffraction can yield low-resolution data that are potentially useful for the characterization of microcrystalline proteins as novel micro- and mesoporous materials as well as for structural studies of biologically important macromolecules.

Features of the secondary structure of a protein molecule from powder diffraction data

Protein powder diffraction is shown to be suitable for obtaining de novo solutions to the phase problem at low resolution via phasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3 A resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality.

Intensity statistics of Friedel opposites.

A previous analysis of the average intensity and mean-square intensity difference of Friedel opposites, confined to the space group P1 [Flack & Shmueli (2007). Acta Cryst. A63, 257-265], is here extended to all the non-centrosymmetric space groups. The present analysis presumes purely non-centrosymmetric content of the unit cell. An important result of this study is that the average intensity and mean-square intensity difference of Friedel opposites have the same values for all the non-centrosymmetric space groups as those previously obtained for the triclinic space group P1. The ratios of average intensity and root-mean-square intensity difference to their triclinic equivalents were derived and exemplified for general as well as for special reflections. For the latter, enhancements were obtained which are shown to be due to those of average intensity and not to a mechanism related to Friedel opposites being explicitly considered.

Polarization-dependence of anomalous scattering in brominated DNA and RNA molecules, and importance of crystal orientation in single- and multiple-wavelength anomalous diffraction phasing

In this paper the anisotropy of anomalous scattering at the Br K-absorption edge in brominated nucleotides is investigated, and it is shown that this effect can give rise to a marked directional dependence of the anomalous signal strength in X-ray diffraction data. This implies that choosing the correct orientation for crystals of such molecules can be a crucial determinant of success or failure when using single- and multiple-wavelength anomalous diffraction (SAD or MAD) methods to solve their structure. In particular, polarized absorption spectra on an oriented crystal of a brominated DNA molecule were measured, and were used to determine the orientation that yields a maximum anomalous signal in the diffraction data. Out of several SAD data sets, only those collected at or near that optimal orientation allowed interpretable electron density maps to be obtained. The findings of this study have implications for instrumental choices in experimental stations at synchrotron beamlines, as well as for the development of data collection strategy programs.

'Broken symmetries' in macromolecular crystallography: phasing from unmerged data.

Site-specific radiation damage and anisotropy of anomalous scattering can induce intensity differences in symmetry-related reflections. If the data are kept unmerged, these symmetry-breaking effects can become a source of phase information.