22 research outputs found
Revealing low-dose radiation damage using single-crystal spectroscopy
Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed
Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography
‘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
Radiation damage in macromolecular crystallography: what is it and why should we care?
The basic causes of the radiation damage inflicted on macromolecular crystals during diffraction experiments are summarized, as well as the current state of research which attempts to understand and to mitigate it
Conformational adaptation of UNCG loops upon crowding
International audienceIf the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are over-represented, the GNRA and the UNCG tetraloops. Although one might think that these consensus sequences imply distinct and specific architectures, such is not the case. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. In this study, crystallographic data derived from both a Lariat-Capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its direct structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly-packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. Importantly, the biological relevance of our observations is supported by the presence of similarly deformed loops in the highly-packed environment of the ribosome and in a complex between a dsRNA and a yeast RNase III. The finding that Z-turn loops can change conformation under higher molecular packing suggests that, in addition to their early demonstrated role in stabilizing RNA folding, they may also contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues
Crystal structure of the aminoglycosides N -acetyltransferase Eis2 from Mycobacterium abscessus
International audienc
Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography
Radiation damage in macromolecular crystals is not suppressed even at 90 K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the `peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an `isosbestic point', each spectrum being a linear combination of `zero' and `infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C-Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging `radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the `isomorphous' signal resulting from its cleavage
Differential modes of peptide binding onto replicative sliding clamps from various bacterial origins
Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.</p