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Yan Zhang, Samuel Hong, Ajchareeya Ruangprasert, Georgios Skiniotis, Christine M Dunham
Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54 Å away from the 30S E site to interact with the 30S head domain and 50S uL5...
February 8, 2018: Structure
Muhammad Ismail, Longbing Ling, Yawei Du, Chen Yao, Xinsong Li
Artemisinin and its derivatives are highly effective drugs in the treatment of P. falciparum malaria. However, their clinical applications face challenges because of short half-life, poor bioavailability and growing drug resistance. In this article, novel dimeric artesunate phospholipid (Di-ART-GPC) based liposomes were developed by combination of dimerization and self-assembly to address these shortcomings. Firstly, Di-ART-GPC conjugate was synthesized by a facile esterification of artesunate (ART) and glycerophosphorylcholine (GPC) and confirmed by MS, 1 H NMR and 13 C NMR...
February 10, 2018: Biomaterials
Sonja-Verena Albers, Ken F Jarrell
Each of the three domains of life exhibits a unique motility structure: while Bacteria use flagella, Eukarya employ cilia, and Archaea swim using archaella. Since the new name for the archaeal motility structure was proposed, in 2012, a significant amount of new data on the regulation of transcription of archaella operons, the structure and function of archaellum subunits, their interactions, and cryo-EM data on in situ archaellum complexes in whole cells have been obtained. These data support the notion that the archaellum is evolutionary and structurally unrelated to the flagellum, but instead is related to archaeal and bacterial type IV pili and emphasize that it is a motility structure unique to the Archaea...
February 13, 2018: Trends in Microbiology
Ke Sherry Li, Liuqing Shi, Michael L Gross
Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure...
February 16, 2018: Accounts of Chemical Research
Daniel F Bogenhagen, Anne G Ostermeyer-Fay, John D Haley, Miguel Garcia-Diaz
Mammalian mtDNA encodes only 13 proteins, all essential components of respiratory complexes, synthesized by mitochondrial ribosomes. Mitoribosomes contain greatly truncated RNAs transcribed from mtDNA, including a structural tRNA in place of 5S RNA as a scaffold for binding 82 nucleus-encoded proteins, mitoribosomal proteins (MRPs). Cryoelectron microscopy (cryo-EM) studies have determined the structure of the mitoribosome, but its mechanism of assembly is unknown. Our SILAC pulse-labeling experiments determine the rates of mitochondrial import of MRPs and their assembly into intact mitoribosomes, providing a basis for distinguishing MRPs that bind at early and late stages in mitoribosome assembly to generate a working model for mitoribosome assembly...
February 13, 2018: Cell Reports
Madoka Akimoto, Bryan VanSchouwen, Giuseppe Melacini
The hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels control nerve impulse transmission and cardiac pacemaker activity. The modulation by cAMP is critical for the regulatory function of HCN in both neurons and cardiomyocytes, but the underlying mechanism is not fully understood. Here, we show how the structure of the apo cAMP-binding domain of the HCN4 isoform has contributed to a model for the cAMP-dependent modulation of the HCN ion-channel. This model recapitulates the structural and dynamical changes that occur along the thermodynamic cycle arising from the coupling of cAMP-binding and HCN self-association equilibria...
February 14, 2018: FEBS Journal
Charlotte A Scarff, Martin J G Fuller, Rebecca F Thompson, Matthew G Iadaza
Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded...
February 6, 2018: Journal of Visualized Experiments: JoVE
Stefan A Arnold, Shirley A Müller, Claudio Schmidli, Anastasia Syntychaki, Luca Rima, Mohamed Chami, H Stahlberg, Kenneth N Goldie, T Braun
This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (EM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies...
February 14, 2018: Proteomics
Amer Alam, Raphael Küng, Julia Kowal, Robert A McLeod, Nina Tremp, Eugenia V Broude, Igor B Roninson, Henning Stahlberg, Kaspar P Locher
The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar...
February 13, 2018: Proceedings of the National Academy of Sciences of the United States of America
Gülsima D Usluer, Frank DiMaio, Shun Kai Yang, Jesse M Hansen, Jessica K Polka, R Dyche Mullins, Justin M Kollman
Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding...
February 13, 2018: Proceedings of the National Academy of Sciences of the United States of America
Shaun Rawson, Claudine Bisson, Daniel L Hurdiss, Asif Fazal, Martin J McPhillie, Svetlana E Sedelnikova, Patrick J Baker, David W Rice, Stephen P Muench
Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies...
February 6, 2018: Proceedings of the National Academy of Sciences of the United States of America
Wen Yang, Ariane Briegel
Cryo-electron microscopy (cryo-EM) allows the imaging of intact macromolecular complexes in the context of whole cells. The biological samples for cryo-EM are kept in a near-native state by flash freezing, without the need for any additional sample preparation or fixation steps. Since transmission electron microscopy only generates 2D projections of the samples, the specimen has to be tilted in order to recover its 3D structural information. This is done by collecting images of the sample with various tilt angles in respect to the electron beam...
2018: Methods in Molecular Biology
Michael D Manson
Like all living organisms, bacteria must communicate with the world around them. As they typically live as single cells, the communication with their environment must occur at the cell membrane, both in moving molecules in and out and in transmitting information about their surroundings to response elements within the cell. This volume is devoted primarily to methods used to study either the behavior of bacteria in response to their environment or methods used to study events that involve signaling pathways that are initiated by events at the cell membrane...
2018: Methods in Molecular Biology
Peter S Shen
The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution." This feature article summarizes some of the major achievements leading to the development of cryo-EM and recent technological breakthroughs that have transformed the method into a mainstream tool for structure determination.
February 9, 2018: Analytical and Bioanalytical Chemistry
Linas Urnavicius, Clinton K Lau, Mohamed M Elshenawy, Edgar Morales-Rios, Carina Motz, Ahmet Yildiz, Andrew P Carter
Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor...
February 7, 2018: Nature
Kristin N Parent, Jason R Schrad, Gino Cingolani
The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell's icosahedral symmetry, providing a gateway through the capsid...
February 7, 2018: Viruses
Kuan Hu, Jordan B Jastrab, Susan Zhang, Amanda Kovach, Gongpu Zhao, K Heran Darwin, Huilin Li
In all domains of life, proteasomes are gated, chambered proteases that require opening by activators to facilitate protein degradation. Twelve proteasome accessory factor E (PafE) monomers assemble into a single dodecameric ring that promotes proteolysis required for the full virulence of the human bacterial pathogen Mycobacterium tuberculosis. Whereas the best characterized proteasome activators use ATP to deliver proteins into a proteasome, PafE does not require ATP. Here, to unravel the mechanism of PafE-mediated protein targeting and proteasome activation, we studied the interactions of PafE with native substrates, including a newly identified proteasome substrate, the ParA-like protein, Rv3213c, and with proteasome core particles...
February 5, 2018: Journal of Biological Chemistry
Sandip Basak, Yvonne Gicheru, Amrita Samanta, Sudheer Kumar Molugu, Wei Huang, Maria la de Fuente, Taylor Hughes, Derek J Taylor, Marvin T Nieman, Vera Moiseenkova-Bell, Sudha Chakrapani
Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4...
February 6, 2018: Nature Communications
Kenta Okamoto, Naoyuki Miyazaki, Hemanth K N Reddy, Max F Hantke, Filipe R N C Maia, Daniel S D Larsson, Chantal Abergel, Jean-Michel Claverie, Janos Hajdu, Kazuyoshi Murata, Martin Svenda
Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer...
February 5, 2018: Virology
Yunxiang Zang, Huping Wang, Zhicheng Cui, Mingliang Jin, Caixuan Liu, Wenyu Han, Yanxing Wang, Yao Cong
Unambiguous subunit assignment in a multicomponent complex is critical for thorough understanding of the machinery and its functionality. The eukaryotic group II chaperonin TRiC/CCT folds approximately 10% of cytosolic proteins and is important for the maintenance of cellular homeostasis. TRiC consists of two rings and each ring has eight homologous but distinct subunits. Unambiguous subunit identification of a macromolecular machine such as TRiC through intermediate or low-resolution cryo-EM map remains challenging...
February 5, 2018: Scientific Reports
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