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cryo EM

Gyu Rie Lee, Lim Heo, Chaok Seok
Advances in protein model refinement techniques are required as diverse sources of protein structure information are available from low-resolution experiments or informatics-based computations such as cryo-EM, NMR, homology models, or predicted residue contacts. Given semi-reliable or incomplete structural information, structure quality of a protein model has to be improved by ab initio methods such as energy-based simulation. In this study, we describe a new automatic refinement server method designed to improve locally inaccurate regions and overall structure simultaneously...
October 16, 2017: Proteins
Christopher Hernandez, Sahil Gulati, Gabriella Fioravanti, Phoebe L Stewart, Agata A Exner
Gas microbubbles stabilized with lipids, surfactants, proteins and/or polymers are widely used clinically as ultrasound contrast agents. Because of their large 1-10 µm size, applications of microbubbles are confined to the blood vessels. Accordingly, there is much interest in generating nanoscale echogenic bubbles (nanobubbles), which can enable new uses of ultrasound contrast agents in molecular imaging and drug delivery, particularly for cancer applications. While the interactions of microbubbles with ultrasound have been widely investigated, little is known about the activity of nanobubbles under ultrasound exposure...
October 18, 2017: Scientific Reports
Ulrich Baxa
TEM is an important method for the characterization of size and shape of nanoparticles as it can directly visualize single particles and even their inner architecture. Imaging of metal particles in the electron microscope is quite straightforward due to their high density and stable structure, but the structure of soft material nanoparticles, such as liposomes, needs to be preserved for the electron microscope. The best method to visualize liposomes close to their native structure is cryo-electron microscopy, where thin films of suspensions are plunge frozen to create vitrified ice films that can be imaged directly in the electron microscope under liquid nitrogen temperature...
2018: Methods in Molecular Biology
Fengbin Wang, Andrew M Burrage, Sandra Postel, Reece E Clark, Albina Orlova, Eric J Sundberg, Daniel B Kearns, Edward H Egelman
The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B...
October 16, 2017: Nature Communications
Kenta Okamoto, Naoyuki Miyazaki, Chihong Song, Filipe R N C Maia, Hemanth K N Reddy, Chantal Abergel, Jean-Michel Claverie, Janos Hajdu, Martin Svenda, Kazuyoshi Murata
The Pithoviridae giant virus family exhibits the largest viral particle known so far, a prolate spheroid up to 2.5 μm in length and 0.9 μm in diameter. These particles show significant variations in size. Little is known about the structure of the intact virion due to technical limitations with conventional electron cryo-microscopy (cryo-EM) when imaging thick specimens. Here we present the intact structure of the giant Pithovirus sibericum particle at near native conditions using high-voltage electron cryo-tomography (cryo-ET) and energy-filtered cryo-EM...
October 16, 2017: Scientific Reports
Michaela Krausová, David Staněk
Split gene architecture of most human genes requires removal of intervening sequences by mRNA splicing that occurs on large multiprotein complexes called spliceosomes. Mutations compromising several spliceosomal components have been recorded in degenerative syndromes and haematological neoplasia, thereby highlighting the importance of accurate splicing execution in homeostasis of assorted adult tissues. Moreover, insufficient splicing underlies defective development of craniofacial skeleton and upper extremities...
October 13, 2017: Seminars in Cell & Developmental Biology
Atsunori Oshima
Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy...
September 21, 2017: Microscopy
Albert Ng, Dong Si
Cryo-electron microscopy (cryo-EM) is a technique that produces three-dimensional density maps of large protein complexes. This allows for the study of the structure of these proteins. Identifying the secondary structures within proteins is vital to understanding the overall structure and function of the protein. The [Formula: see text]-barrel is one such secondary structure, commonly found in lipocalins and membrane proteins. In this article, we present a novel approach that utilizes genetic algorithms, kd-trees, and ray tracing to automatically detect and extract [Formula: see text]-barrels from cryo-EM density maps...
October 16, 2017: Journal of Computational Biology: a Journal of Computational Molecular Cell Biology
Gregory M Martin, Balamurugan Kandasamy, Frank DiMaio, Craig Yoshioka, Show-Ling Shyng
Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites...
October 16, 2017: ELife
Gabriel Demo, Aviram Rasouly, Nikita Vasilyev, Vladimir Svetlov, Anna B Loveland, Ruben Diaz-Avalos, Nikolaus Grigorieff, Evgeny Nudler, Andrei A Korostelev
In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit...
October 13, 2017: ELife
Soledad Baños-Mateos, Anne-Marie M van Roon, Ulla F Lang, Sarah L Maslen, J Mark Skehel, Meindert H Lamers
High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues...
October 11, 2017: Nature Communications
Annapurna Vemu, Joseph Atherton, Jeffrey O Spector, Carolyn A Moores, Antonina Roll-Mecak
Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4...
October 11, 2017: Molecular Biology of the Cell
Marscha Hirschi, Mark A Herzik, Jinhong Wie, Yang Suo, William F Borschel, Dejian Ren, Gabriel C Lander, Seok-Yong Lee
The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca(2+)-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca(2+) from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype...
October 11, 2017: Nature
Harry B Gristick, Haoqing Wang, Pamela J Bjorkman
The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures...
October 1, 2017: Acta Crystallographica. Section D, Structural Biology
Mayuriben Parmar, Shaun Rawson, Charlotte A Scarff, Adrian Goldman, Timothy R Dafforn, Stephen P Muench, Vincent L G Postis
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs...
October 6, 2017: Biochimica et Biophysica Acta
Mark A Herzik, Mengyu Wu, Gabriel C Lander
Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
October 9, 2017: Nature Methods
Pavel Afanasyev, Charlotte Seer-Linnemayr, Raimond B G Ravelli, Rishi Matadeen, Sacha De Carlo, Bart Alewijnse, Rodrigo V Portugal, Navraj S Pannu, Michael Schatz, Marin van Heel
Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets...
September 1, 2017: IUCrJ
Tai Wei Guo, Alberto Bartesaghi, Hui Yang, Veronica Falconieri, Prashant Rao, Alan Merk, Edward T Eng, Ashleigh M Raczkowski, Tara Fox, Lesley A Earl, Dinshaw J Patel, Sriram Subramaniam
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved...
October 5, 2017: Cell
Sebastian M Fica, Kiyoshi Nagai
The spliceosome excises introns from pre-messenger RNAs using an RNA-based active site that is cradled by a dynamic protein scaffold. A recent revolution in cryo-electron microscopy (cryo-EM) has led to near-atomic-resolution structures of key spliceosome complexes that provide insight into the mechanism of activation, splice site positioning, catalysis, protein rearrangements and ATPase-mediated dynamics of the active site. The cryo-EM structures rationalize decades of observations from genetic and biochemical studies and provide a molecular framework for future functional studies...
October 5, 2017: Nature Structural & Molecular Biology
James A Letts, Leonid A Sazanov
The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI-CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII2 and CIV (SC I+III2+IV, known as the respirasome), as well as with CIII2 alone (SC I+III2)...
October 5, 2017: Nature Structural & Molecular Biology
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