Project description:We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
Project description:We report the determination of the structure of Escherichia coli β-galactosidase at a resolution of ∼1.8 Å using data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. The data were collected in a single 24 h session by recording images from an array of 7 × 7 holes at each stage position using the automated data collection program SerialEM. In addition to the expected features such as holes in the densities of aromatic residues, the map also shows density bumps corresponding to the locations of hydrogen atoms. The hydrogen densities are useful in assigning absolute orientations for residues such as glutamine or asparagine by removing the uncertainty in the fitting of the amide groups, and are likely to be especially relevant in the context of structure-guided drug design. These findings validate the use of electron microscopes operating at 200 kV for imaging protein complexes at atomic resolution using cryo-EM.
Project description:The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ?-galactosidase bound to the inhibitor phenylethyl ?-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ? 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.
Project description:Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.
Project description:Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer's disease patients. We present the structure of an Aβ(1-42) fibril composed of two intertwined protofilaments determined by cryo-electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-β structure resulting in an overall "LS"-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed "groove" and "ridge," leading to different binding pathways on both fibril ends, which has implications for fibril growth.