Non-rigid image registration to reduce beam-induced blurring of cryo-electron microscopy images

Microscopic imaging and technolog

Radiation damage to nucleoprotein complexes in macromolecular crystallography

Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein-DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07-44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1-C and sugar-phosphate C-O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses

Iridium-catalysed synthesis of C, N, N -cyclic azomethine imines enables entry to unexplored nitrogen-rich 3D chemical space

Three-dimensional nitrogen-rich bridged ring systems are of great interest in drug discovery owing to their distinctive physicochemical and structural properties. However, synthetic approaches towards N–N-bond-containing bridged heterocycles are often inefficient and require tedious synthetic strategies. Here we delineate an iridium-catalysed reductive approach to such architectures from C, N, N-cyclic hydrazide substrates using IrCl(CO)[P(OPh)3]2 and 1,1,3,3-tetramethyldisiloxane (TMDS), which provided efficient access to the unstabilized and highly reactive C, N, N-cyclic azomethine imine dipoles. These species were stable and isolable in their dimeric form, but, upon dissociation in solution, reacted with a broad range of dipolarophiles in [3 + 2] cycloaddition reactions with high yields and good diastereoselectivities, enabling the direct synthesis of nitrogen-rich sp3-hybridized pyrazoline polycyclic ring systems. Density functional theory calculations were performed to elucidate the origin of the diastereoselectivity of the cycloaddition reaction, and principal moment of inertia (PMI) analysis was conducted to enable visualization of the topological information of the dipolar cycloadducts

RNA protects a nucleoprotein complex against radiation damage

Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3-25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein-DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density

The molecular basis for apolipoprotein E4 as the major risk factor for late onset Alzheimer's disease

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an -helical, 299-amino acid protein. Homozygosity for the ε4 allele is the major risk factor for developing late onset Alzheimer’s disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158 and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerisation state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small angle X-ray scattering, analytical ultracentrifugation, circular dichroism, X-ray fibre diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerise to form wavy filaments which do not share the characteristics of cross- amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis and these results open opportunities for uncovering new triggers for AD onset