We propose a definition of local resolution for three-dimensional electron cryo-microscopy (cryo-EM) density maps that uses local sinusoidal features. Our algorithm has no free parameters and is applicable to other imaging modalities, including tomography. By evaluating the local resolution of single-particle reconstructions and subtomogram averages for four example data sets, we report variable resolution across a 4- to 40-A range.

/pdf/quantifying-the-local-resolution-of-cryo-em-density-maps-194ktl42w0.pdf

Quantifying the local resolution of cryo-EM density maps.

Stress-signaling pathways are evolutionarily conserved and play an important role in the maintenance of homeostasis. These pathways are also critical for adaptation to new cellular environments. The endoplasmic reticulum (ER) unfolded protein response (UPR) is activated by biosynthetic stress and leads to a compensatory increase in ER function. The JNK and p38 MAPK signaling pathways control adaptive responses to intracellular and extracellular stresses, including environmental changes such as UV light, heat, and hyperosmotic conditions, and exposure to inflammatory cytokines. Metabolic stress caused by a high-fat diet represents an example of a stimulus that coordinately activates both the UPR and JNK/p38 signaling pathways. Chronic activation of these stress-response pathways ultimately causes metabolic changes associated with obesity and altered insulin sensitivity. Stress-signaling pathways, therefore, represent potential targets for therapeutic intervention in the metabolic stress response and other disease processes.

/pdf/cell-signaling-and-stress-responses-3gujnsaem8.pdf

Cell Signaling and Stress Responses

One number does not fit all: Mapping local variations in resolution in cryo-EM reconstructions

The WHO recognizes human African trypanosomiasis, Chagas disease and the leishmaniases as neglected tropical diseases. These diseases are caused by parasitic trypanosomatids and range in severity from mild and self-curing to near invariably fatal. Public health advances have substantially decreased the effect of these diseases in recent decades but alone will not eliminate them. In this Review, we discuss why new drugs against trypanosomatids are required, approaches that are under investigation to develop new drugs and why the drug discovery pipeline remains essentially unfilled. In addition, we consider the important challenges to drug discovery strategies and the new technologies that can address them. The combination of new drugs, new technologies and public health initiatives is essential for the management, and hopefully eventual elimination, of trypanosomatid diseases from the human population.

/pdf/anti-trypanosomatid-drug-discovery-an-ongoing-challenge-and-2ekqaaeynk.pdf

Anti-trypanosomatid drug discovery: an ongoing challenge and a continuing need

Cellular electron cryotomography offers researchers the ability to observe macromolecules frozen in action in situ, but a primary challenge with this technique is identifying molecular components within the crowded cellular environment. We introduce a method that uses neural networks to dramatically reduce the time and human effort required for subcellular annotation and feature extraction. Subsequent subtomogram classification and averaging yield in situ structures of molecular components of interest. The method is available in the EMAN2.2 software package.

/pdf/convolutional-neural-networks-for-automated-annotation-of-4ia0eaeqmt.pdf

Convolutional neural networks for automated annotation of cellular cryo-electron tomograms

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

/pdf/hepatitis-c-virus-like-internal-ribosome-entry-sites-4lxm68tv6v.pdf

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit.

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.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.

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Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.