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Van Son Nguyen, Badreddine Douzi, Eric Durand, Alain Roussel, Eric Cascales, Christian Cambillau
The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM...
February 1, 2018: Current Opinion in Structural Biology
Christopher J Russo, Richard Henderson
The fluctuating granularity or "bee swarm" effect seen in highly defocussed transmission electron micrographs is caused by microscopic charge fluctuations in the specimen created by the illuminating beam. In the field of high-resolution single particle electron cryomicroscopy (cryoEM), there has been a concern that this fluctuating charge might cause defocus-dependent Thon ring fading which would degrade the final image. In this paper, we have analysed the 2.35 Å fringes from the (111) reflection in images of gold nanoparticles embedded in amorphous ice...
January 31, 2018: Ultramicroscopy
Christopher J Russo, Richard Henderson
When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work...
January 31, 2018: Ultramicroscopy
Fuxing Zeng, Hong Jin
The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control...
February 5, 2018: Scientific Reports
Venkata P Dandey, Hui Wei, Zhening Zhang, Yong Zi Tan, Priyamvada Acharya, Edward T Eng, William J Rice, Peter A Kahn, Clinton S Potter, Bridget Carragher
We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.
January 20, 2018: Journal of Structural Biology
Hui Wei, Venkata P Dandey, Zhening Zhang, Ashleigh Raczkowski, Willam J Rice, Bridget Carragher, Clinton S Potter
We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes...
January 6, 2018: Journal of Structural Biology
Pascal Krotee, Sarah L Griner, Michael R Sawaya, Duilio Cascio, Jose A Rodriguez, Dan Shi, Stephan Philipp, Kevin Murray, Lorena Saelices, Ji Lee, Paul Seidler, Charles G Glabe, Lin Jiang, Tamir Gonen, David S Eisenberg
Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues, and are associated with Alzheimer's disease (AD) and Type-II Diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method Micro-Electron Diffraction (MicroED) we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ 24-34 WT and hIAPP 19-29 S20G, with 64% sequence similarity...
December 27, 2017: Journal of Biological Chemistry
Elena E Grintsevich, Peng Ge, Michael R Sawaya, Hunkar Gizem Yesilyurt, Jonathan R Terman, Z Hong Zhou, Emil Reisler
Actin filament assembly and disassembly are vital for cell functions. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin's M44 and M47 residues to induce cellular F-actin disassembly. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin's nucleotide-bound state. Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin...
December 19, 2017: Nature Communications
Marie C Stark, Mo A Baikoghli, Tanja Lahtinen, Sami Malola, Li Xing, Michelle Nguyen, Marina Nguyen, Aria Sikaroudi, Varpu Marjomäki, Hannu Häkkinen, R Holland Cheng
Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core...
December 6, 2017: Scientific Reports
Shiheng Liu, Xueni Li, Lingdi Zhang, Jiansen Jiang, Ryan C Hill, Yanxiang Cui, Kirk C Hansen, Z Hong Zhou, Rui Zhao
The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, B(act), C, C*, and ILS complexes revealed mechanisms of 5' splice site (ss) recognition, branching, and intron release, but lacked information on 3' ss recognition, exon ligation and release. Here, we report a cryoEM structure of the postcatalytic P complex at 3.3Å resolution, revealing that 3' ss is mainly recognized through non-Watson-Crick basepairing with the 5' ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3' exon and potentially regulating Prp22 activity...
November 16, 2017: Science
C Keith Cassidy, Benjamin A Himes, Zaida Luthey-Schulten, Peijun Zhang
Recent advances in cryo-electron microscopy (cryoEM) have dramatically improved the resolutions at which vitrified biological specimens can be studied, revealing new structural and mechanistic insights over a broad range of spatial scales. Bolstered by these advances, much effort has been directed toward the development of hybrid modeling methodologies for the construction and refinement of high-fidelity atomistic models from cryoEM data. In this brief review, we will survey the key elements of cryoEM-based hybrid modeling, providing an overview of available computational tools and strategies as well as several recent applications...
November 3, 2017: Current Opinion in Microbiology
Carolyn Bertozzi
No abstract text is available yet for this article.
October 25, 2017: ACS Central Science
Philip R Baldwin, Yong Zi Tan, Edward T Eng, William J Rice, Alex J Noble, Carl J Negro, Michael A Cianfrocco, Clinton S Potter, Bridget Carragher
The scope and complexity of cryogenic electron microscopy (cryoEM) data has greatly increased, and will continue to do so, due to recent and ongoing technical breakthroughs that have led to much improved resolutions for macromolecular structures solved using this method. This big data explosion includes single particle data as well as tomographic tilt series, both generally acquired as direct detector movies of ∼10-100 frames per image or per tilt-series. We provide a brief survey of the developments leading to the current status, and describe existing cryoEM pipelines, with an emphasis on the scope of data acquisition, methods for automation, and use of cloud storage and computing...
October 31, 2017: Current Opinion in Microbiology
Alexandra C Walls, M Alejandra Tortorici, Joost Snijder, Xiaoli Xiong, Berend-Jan Bosch, Felix A Rey, David Veesler
The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine...
October 17, 2017: Proceedings of the National Academy of Sciences of the United States of America
Christopher J Williams, Jeffrey J Headd, Nigel W Moriarty, Michael G Prisant, Lizbeth L Videau, Lindsay N Deis, Vishal Verma, Daniel A Keedy, Bradley J Hintze, Vincent B Chen, Swati Jain, Steven M Lewis, W Bryan Arendall, Jack Snoeyink, Paul D Adams, Simon C Lovell, Jane S Richardson, David C Richardson
This paper describes the current update on macromolecular model validation services that are provided at the MolProbity website, emphasizing changes and additions since the previous review in 2010. There have been many infrastructure improvements, including rewrite of previous Java utilities to now use existing or newly written Python utilities in the open-source CCTBX portion of the Phenix software system. This improves long-term maintainability and enhances the thorough integration of MolProbity-style validation within Phenix...
October 25, 2017: Protein Science: a Publication of the Protein Society
Xueni Li, Shiheng Liu, Jiansen Jiang, Lingdi Zhang, Sara Espinosa, Ryan C Hill, Kirk C Hansen, Z Hong Zhou, Rui Zhao
U1 snRNP plays a critical role in 5'-splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. Here, we report the cryoEM structure of yeast U1 snRNP at 3.6 Å resolution with atomic models for ten core proteins, nearly all essential domains of its RNA, and five stably associated auxiliary proteins...
October 19, 2017: Nature Communications
Chaity Banerjee, Zhongjun Hu, Zhong Huang, J Anthony Warrington, Dianne W Taylor, Kathleen M Trybus, Susan Lowey, Kenneth A Taylor
Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops...
October 14, 2017: Journal of Structural Biology
Yun-Tao Liu, Jiansen Jiang, Kevin Patrick Bohannon, Xinghong Dai, G W Gant Luxton, Wong Hoi Hui, Guo-Qiang Bi, Gregory Allan Smith, Z Hong Zhou
Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM)...
November 2017: Journal of General Virology
Xinghong Dai, Lily Wu, Ren Sun, Z Hong Zhou
Human adenoviruses (Ad) are dsDNA viruses associated with infectious diseases, yet better known as tools for gene delivery and oncolytic anti-cancer therapy. Atomic structures of Ad provide the basis for the development of antivirals and for engineering efforts towards more effective applications. Since 2010, atomic models of human Ad5 have been independently derived from photographic film cryoEM and X-ray crystallography, but discrepancies exist concerning the assignment of cement proteins IIIa, VIII and IX...
October 4, 2017: Journal of Virology
Miguel Zamora, Eduardo Méndez-López, Xabier Agirrezabala, Rebeca Cuesta, José L Lavín, M Amelia Sánchez-Pina, Miguel A Aranda, Mikel Valle
Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å...
September 2017: Science Advances
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