Abstract
In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently ...computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC–STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC–STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.
Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been ...shown to possess various spatial conformations, including a ring-shaped configuration which was considered to be functionally important. However, a recent in situ cryo-ET analysis of predominant regular inter-ribosome contacts did not confirm the abundance of ring-shaped polyribosomes in a cell cytoplasm. To address this discrepancy, here we analyzed the cryo-ET structure of polyribosomes in diluted lysates of HeLa cells. It was shown that the vast majority of the ribosomes were combined into polysomes and were proven to be translationally active. Tomogram analysis revealed that circular polyribosomes are indeed very common in the cytoplasm, but they mostly possess pseudo-regular structures without specific inter-ribosomal contacts. Although the size of polyribosomes varied widely, most circular polysomes were relatively small in size (4-8 ribosomes). Our results confirm the recent data that it is cellular mRNAs with short ORF that most commonly form circular structures providing an enhancement of translation.
The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical ...information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years
. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA
receptor homopentamer
. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
Plant viruses and their virus-like particles (VLPs) have a lot of advantages for biotechnological applications including complete safety for humans. Alternanthera mosaic virus (AltMV) is a ...potentially promising object for design of novel materials. The 3D structures of AltMV virions and its VLPs were obtained by single particle EM at ~13Å resolution. The comparison of the reconstructions and a trypsin treatment revealed that AltMV CPs possesses a different fold in the presence (virions) and absence of viral RNA (VLPs). For the first time, the structure of morphologically similar virions and virus-like particles based on the coat protein of a helical filamentous plant virus is shown to be different. Despite this, both AltMV virions and VLPs are stable in a wide range of conditions. To provide a large amount of AltMV for biotechnology usage the isolation procedure was modified.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
N-terminally substituted lysine derivatives of gramicidin A (gA), Lys1gA and Lys3gA, but not glutamate- or aspartate-substituted peptides have been previously shown to cause the leakage of ...carboxyfluorescein from liposomes. Here, the leakage induction was also observed for Arg1gA and Arg3gA, while His1gA and His3gA were inactive at neutral pH. The Lys3-containing analogue with all tryptophans replaced by isoleucines did not induce liposome leakage, similar to gA. This suggests that the presence of both tryptophans and N-terminal cationic residues is critical for pore formation. Remarkably, the addition of gA blocked the leakage induced by Lys3gA. By examining with fluorescence correlation spectroscopy the peptide-induced leakage of fluorescent markers from liposomes, we estimated the diameter of pores responsible for the leakage to be about 1.6 nm. Transmission electron cryo-microscopy imaging of liposomes with Lys3gA showed that the liposomal membranes contained high electron density particles with a size of about 40 Å, suggesting the formation of peptide clusters. No such clusterization was observed in liposomes incorporating gA or a mixture of gA with Lys3gA. Three-dimensional reconstruction of the clusters was compatible with their pentameric arrangement. Based on experimental data and computational modeling, we suggest that the large pore formed by Lys3gA represents a barrel-stave oligomeric cluster formed by antiparallel double-stranded helical dimers (DH). In a tentative model, the pentamer of dimers may be stabilized by aromatic Trp-Trp and cation-π Trp-Lys interactions between the neighboring DHs. The inhibiting effect of gA on the Lys3gA-induced leakage can be attributed to breaking of cation-π interactions, which prevents peptide clusterization and pore formation.
Gramicidin A (gA) blocks dye leakage from liposomes induced by Lys3gA, thereby highlighting the importance of cation-π interactions for pore formation. Based on cryo-em, large pores are attributed to 40 Å-diameter peptide clusters.
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Amphiphilic copolymers of N-vinylpyrrolidone and triethylene glycol dimethacrylate, synthesized by radical copolymerization at equimolar and unequimolar brancher and chain transfer ...agent ratios, as well as their precipitated fractions with given chemical composition and physico-chemical characteristics were applied to produce the new stable nanoscaled macromolecular hybrid structures of C60 fullerene through spontaneous aggregation of copolymer particles in isopropyl alcohol and C60 molecules dissolved in toluene. FTIR and electronic absorption spectroscopy data indicate the formation of the macromolecular complex of “guest-host” as a result of the C60 solubilization by the polymer particles. The electron structure of fullerene was changed rapidly or slowly in this complex due to donor–acceptor interaction depending on copolymer kind in the solution. According to WAXS, the encapsulated fullerene molecularly dispersed in solid copolymer matrix. The dynamic characteristics (diffusion coefficients Dt and hydrodynamic radii Rh) of hybrid macromolecular structures in polar media (isopropyl alcohol, water) were determined. Cryo-TEM image shows the core–shell particles of hybrid macromolecular structure; the fullerene particles of about 3nm size were visible in the core of micellar particles.
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