Project description:Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.
Project description:The crystal structure of a 40 kDa signalling glycoprotein from buffalo (SPB-40) has been determined at 2.8 A resolution. SPB-40 acts as a protective signalling factor by binding to viable cells during the early phase of involution, during which extensive tissue remodelling occurs. It was isolated from the dry secretions of Murrah buffalo. It was purified and crystallized using the hanging-drop vapour-diffusion method with 19% ethanol as the precipitant. The protein was also cloned and its complete nucleotide and amino-acid sequences were determined. When compared with the sequences of other members of the family, the sequence of SPB-40 revealed two very important mutations in the sugar-binding region, in which Tyr120 changed to Trp120 and Glu269 changed to Trp269. The structure showed a significant distortion in the shape of the sugar-binding groove. The water structure in the groove is also drastically altered. The folding of the protein chain in the flexible region comprising segments His188-His197, Phe202-Arg212 and Tyr244-Pro260 shows large variations when compared with other proteins of the family.