The methods for treating experimental data in the isomorphous replacement and anomalous scattering methods of macromolecular phase determination have undergone considerable evolution since their inception 50 years ago. The successive formulations used are reviewed, from the most simplistic viewpoint to the most advanced, including the exploration of some blind alleys. A new treatment is proposed and demonstrated for the improved encoding and subsequent exploitation of phase information in the complex plane. It is concluded that there is still considerable scope for further improvements in the statistical analysis of phase information, which touch upon numerous fundamental issues related to data processing and experimental design.

X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination.

The gene coding for urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin, is inactivated in humans. Consequently, urate oxidase is used as a protein drug to overcome severe disorders induced by uric acid accumulation. The structure of the active homotetrameric enzyme reveals the existence of a small architectural domain that we call T-fold (for tunnelling-fold) domain. It assembles to form a perfect unusual dimeric alpha 8 beta 16 barrel. Urate oxidase may be the archetype of an expanding new family of tunnel-shaped proteins that now has three members; tetrahydropterin synthase, GTP cyclohydrolase I and urate oxidase. The structure of the active site of urate oxidase around the 8-azaxanthine inhibitor reveals an original mechanism of oxidation that does not require any ions or prosthetic groups.

A well‐defined structural phase transformation is observed in bulk single crystals of pentacene, whereas pentacene powders heated above the phase‐transformation temperature do not always fully convert, and upon cooling, coexistence of the two polymorphs is observed down to room temperature. The first‐order phase transformation is isostructural: the close‐packed herringbone‐type layers shift against each other, keeping the same symmetry.

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 higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by 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 α 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.T he specific labeling of proteins with synthetic probes is a powerful approach for studying protein function. One way to achieve such a specific labeling is based on so-called selflabeling protein tags.1 In this approach, the protein of interest is expressed as a fusion protein with a peptide or protein (i.e., tag) whose role is to specifically bind to a synthetic probe in vitro or in vivo. A well-established example of a self-labeling protein tag is SNAP-tag.2 SNAP-tag specifically reacts with substituted O 6 -benzylguanine derivatives and thereby permits the labeling of SNAP-tag fusion proteins with a wide variety of different synthetic probes. Recent applications include its use for the analysis of protein complexes, 3 super-resolution microscopy, 4 the identification of protein−protein interactions, 5 drug target identification, 6 and the determination of protein half-life in animals. 7 The appeal of self-labeling tags such as SNAP-tag is the ease with which fusion proteins can be labeled with synthetic probes even in living cells. A conceptual limitation of the approach is the fact that the tag can affect the properties of its fusion partner. It is therefore important that the properties of the tag be as thoroughly characterized as possible.SNAP-tag was generated in a stepwise manner from human O 6 -alkylguanine-DNA alkyltransferase (hAGT) by introduction of a total of 19 point mutations (Figure 1) and deletion of 25 C-terminal residues. Saturation mutagenesis of four active-site residues followed by phage display and selection for activity against BG derivatives resulted in GE AGT, a mutant with 20-fold increased activity toward such substrates ( Figure 1B). 8Subsequent saturation mutagenesis of four additional residues involved in substrate binding followed by phage selection resulted in AGT-54, a mutant with 1.5-fold higher activity than GE AGT. To further optimize the protein for applications in protein labeling, mutations were introduced to suppress DNA binding and reactivity toward nucleosides, to remove nonessential cysteines, and to truncate the last 25 residues. 9The resulting mutant M AGT displayed relatively low activ...

Recently, there has been a resurgence in phasing using the single-wavelength anomalous diffraction (SAD) experiment -data from a single wavelength in combination with density modification techniques have been used to solve structures, even with a very small anomalous signal. Furthermore, SAD can be favorable to a multi-wavelength anomalous diffraction (MAD) experiment in a case where a crystal exhibits radiation decay during the course of a MAD experiment.Currently, to refine the anomalous substructure and phase a SAD data set, conventional techniques employ a least squares function either on the Bijvoet differences or the Bijvoet/Friedal pairs. These equations neglect some of the important correlations that occur in a SAD experiment. Indeed, since data from a SAD experiment comes from the same crystal and share the same model of anomalous scatterers, explicitly accounting for these correlations may improve results further. Here, a novel formulation for SAD phasing and refinement employing multivariate statistical techniques is presented. The equation developed accounts explicitly for the correlations from the observed and calculated Friedal mates in a SAD experiment. Furthermore, the function derived requires only a one dimensional numerical integration. The correlated SAD equation has been implemented and test cases performed on real diffraction data have revealed significantly better results than the currently most used programs in terms of correlation with the final map and producing more reliable phase probability statistics. We present the largest successful application of selenomethionine MAD reported to date: the crystal structure of the decameric E.coli enzyme ketopantoate hydroxymethyltransferase (KPHMT), with 160 ordered selenium atoms and 560 kDa of protein in the asymmetric unit. Despite small (<150 µ m), irregular, weakly diffracting (<3.2 Å) crystals, the substructure was solved by SAD combined with Direct Methods, using a 20-fold redundant "peak" dataset. SnB 1 produced the first correct solution after 2600 computing hours, and phases from SHARP 2 and Solomon 3 produced traceable maps, even before 20-fold NCS averaging. Subsequent analysis revealed that data redundancy was critical for success; on the other hand, speed and success rate vary considerably between direct methods programs. Apart from a favorable ratio of selenium to scattering matter, the procedure was quite general, suggesting that this is still a long way from the practical upper limit of applicability, if that exists. The current release (1.4.0) of SHARP [1] has been incorporated into a set of scripts called 'autoSHARP' which extend in both the upstream and downstream directions the initial coupling of SHARP to the SOLOMON density modification program. Upstream, autoSHARP can perform data checking and scaling, and heavy-atom detection; downstream, it can carry out density modification with automatic optimization of solvent flattening parameter and choice of hand, and launch the ARP/wARP map interpretation and model building proto...

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 K͒ 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.

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.