Xiao Chen Bai

Cellular internalization of exosomes occurs through phagocytosis

Exosomes play important roles in many physiological and pathological processes. However, the exosome–cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non‐phagocytic cells. Most exosomes were observed attached to the plasma membrane of non‐phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate‐modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3‐kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant‐negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin‐coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome–cell interactions and the exosome intracellular trafficking pathway.

Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens. DOI: http://dx.doi.org/10.7554/eLife.00461.001

How cryo-EM is revolutionizing structural biology

For many years, structure determination of biological macromolecules by cryo-electron microscopy (cryo-EM) was limited to large complexes or low-resolution models. With recent advances in electron detection and image processing, the resolution by cryo-EM is now beginning to rival X-ray crystallography. A new generation of electron detectors record images with unprecedented quality, while new image-processing tools correct for sample movements and classify images according to different structural states. Combined, these advances yield density maps with sufficient detail to deduce the atomic structure for a range of specimens. Here, we review the recent advances and illustrate the exciting new opportunities that they offer to structural biology research.

Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution.

The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation–contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8xa0Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

An atomic structure of human γ-secretase

Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer’s disease, with most mutations derived from Alzheimer’s disease mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Mutations derived from Alzheimer’s disease affect residues at two hotspots in PS1, each located at the centre of a distinct four transmembrane segment (TM) bundle. TM2 and, to a lesser extent, TM6 exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain after substrate recruitment. Component protein APH-1 serves as a scaffold, anchoring the lone transmembrane helix from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.

Three-dimensional structure of human γ-secretase

The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer’s disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 Å resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex.

Sampling the conformational space of the catalytic subunit of human γ-secretase

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimers disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain. DOI: http://dx.doi.org/10.7554/eLife.11182.001

Structure of the large ribosomal subunit from human mitochondria

Making mitochondrial hydrophobic proteins Mitochondria produce chemical energy for the cell. Human mitochondria have their own specific ribosomes—mitoribosomes, which are distinct from cytoplasmic ribosomes. Mitoribosomes synthesize the mitochondrial membrane proteins that generate the chemical energy. Brown et al. used cryo–electron microscopy to determine the high-resolution structure of the large subunit of the human mitoribosome. The mitoribosome has a number of unique features, including an exit tunnel lined with hydrophobic amino acid residues. Science, this issue p. 718 The structure of the human mitochondrial ribosome reveals how it is adapted to synthesize mitochondrial membrane proteins. Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNAVal) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

Cryo-EM structure of a 3D DNA-origami object

A key goal for nanotechnology is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromolecular complexes. Molecular self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoatomic model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnology and experimental evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that observed in natural macromolecules. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback.

Initiation of Translation by Cricket Paralysis Virus IRES Requires Its Translocation in the Ribosome

Summary The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.