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

Instrument-independent specification of the diffraction geometry and polarization state of the incident X-ray beam

This work augments the proposal of Schwarzenbach & Flack [J. Appl. Cryst. (1989), 22, 601-605], who have advocated the use of a diffractometer-independent definition of the azimuthal angle $\psi$ to specify the diffractiongeometry of a Bragg reflection. It is here proposed that one additional angle $\xi$, which is also based on a diffractometer-independent definition, is needed to encode the direction of linear polarization for those experiments where this quantity is of importance. This definition is then extended to the cases of partially and/or elliptically polarized X-ray beams, and the use of three normalized Stokes parameters, P$_1$, P$_2$ and P$_3$, together with $\xi$, is advocated in order to characterize exhaustively the polarization state of the incident beam. The conventions proposed here present a general, unambiguous and economical means of encoding the information about the diffraction geometry, without the need to record any further information about the instrument, crystal orientation matrix and goniometer angles. Data-processing software using these definitions to analyse polarization-dependent phenomena becomes instrument-independent and completely general. These methods have been implemented in the macromolecular phasing program SHARP for exploiting the polarization anisotropy of anomalous scattering in protein crystals

‘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

Preliminary crystallographic analysis of two oligomerization-deficient mutants of the aerolysin toxin, H132D and H132N, in their proteolyzed forms

Aerolysin is a major virulence factor produced by the Gram-negative bacterium Aeromonas hydrophila and is a member of the beta-pore-forming toxin family. Two oligomerization-deficient aerolysin mutants, H132D and H132N, have been overproduced, proteolyzed by trypsin digestion and purified. Crystals were grown from the proteolyzed forms and diffraction data were collected for the two mutants to 2.1 and 2.3 A resolution, respectively. The prism-shaped crystals belonged to space group C2. The crystal structure of the mutants in the mature, but not heptameric, aerolysin form will provide insight into the intermediate states in the oligomerization process of a pore-forming toxin

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 heavyatom 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

Directed evolution of the suicide protein O⁶-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to that the reactivity of the suicide enzyme can be influenced by higher stability, especially of the alkylated protein, suggesting stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key a helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein

Crystal Structure of the PP2A Phosphatase Activator: Implications for Its PP2A-Specific PPIase Activity

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all α-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity

Molecular envelopes derived from protein powder diffraction 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