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Single particle electron microscopy

Kaustuv Basu, Driss Lajoie, Tristan Aumentado-Armstrong, Jin Chen, Roman I Koning, Blaise Bossy, Mihnea Bostina, Attila Sik, Ella Bossy-Wetzel, Isabelle Rouiller
Mitochondria are dynamic organelles that continually adapt their morphology by fusion and fission events. An imbalance between fusion and fission has been linked to major neurodegenerative diseases, including Huntington's, Alzheimer's, and Parkinson's diseases. A member of the Dynamin superfamily, dynamin-related protein 1 (DRP1), a dynamin-related GTPase, is required for mitochondrial membrane fission. Self-assembly of DRP1 into oligomers in a GTP-dependent manner likely drives the division process. We show here that DRP1 self-assembles in two ways: i) in the presence of the non-hydrolysable GTP analog GMP-PNP into spiral-like structures of ~36 nm diameter; and ii) in the presence of GTP into rings composed of 13-18 monomers...
2017: PloS One
Joachim Frank
Time-resolved cryo-electron microscopy (cryo-EM) combines the known advantages of single-particle cryo-EM in visualizing molecular structure with the ability to dissect the time progress of a reaction between molecules in vitro. Here some of the recent progress of this methodology and its first biological applications are outlined.
June 15, 2017: Journal of Structural Biology
Yan Han, Chunli Yan, Thi Hoang Duong Nguyen, Ashleigh J Jackobel, Ivaylo Ivanov, Bruce A Knutson, Yuan He
Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from -27 to -16. Core Factor's intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location...
June 17, 2017: ELife
Lisa Nika, Jakob Wallner, Dieter Palmberger, Krisztina Koczka, Karola Vorauer-Uhl, Reingard Grabherr
Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins...
June 12, 2017: Protein Expression and Purification
Luisa Campagnolo, Micol Massimiani, Lucia Vecchione, Diletta Piccirilli, Nicola Toschi, Andrea Magrini, Elena Bonanno, Manuel Scimeca, Luca Castagnozzi, Giorgio Buonanno, Luca Stabile, Francesco Cubadda, Federica Aureli, Paul Hb Fokkens, Wolfgang G Kreyling, Flemming R Cassee, Antonio Pietroiusti
Recently, interest for the potential impact of consumer-relevant engineered nanoparticles on pregnancy has dramatically increased. This study investigates whether inhaled silver nanoparticles (AgNPs) reach and cross mouse placental barrier and induce adverse effects. Apart from their relevance for the growing use in consumer products and biomedical applications, AgNPs are selected since they can be unequivocally identified in tissues. Pregnant mouse females are exposed during the first 15 days of gestation by nose-only inhalation to a freshly produced aerosol of 18-20 nm AgNPs for either 1 or 4 hours, at a particle number concentration of 3...
June 16, 2017: Nanotoxicology
Lin Wei, Yanhong Ma, Xupeng Zhu, Jianghong Xu, Yaxin Wang, Huigao Duan, Lehui Xiao
In this work, with wavelength-resolved dark-field microscopy, the center-of-mass localization information from nanoparticle pairs (i.e., spherical (45 nm in diameter) and rod (45 × 70 nm) shaped gold nanoparticle pairs with different gap distances and orientations) was explored and compared with the results determined by scanning electron microscopy (SEM) measurements. When the gap distance was less than 20 nm, the scattering spectrum of the nanoparticle pair was seriously modulated by the plasmonic coupling effect...
June 15, 2017: Nanoscale
Rameez Ahmad Mir, Piyush Sharma, Om Prakash Pandey
Carbon coated nano molybdenum carbide (Mo2C) has been synthesized at 800 °C through single step reduction route using molybdenum trioxide (MoO3) as a precursor, polypropylene (P.P) as a carbon source and magnesium (Mg) as a catalyst in an autoclave. The synthesized samples were characterized by X-ray diffraction (XRD), thermal analysis techniques (TG/DTA/DTG), field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Williamson- Hall (W-H) analysis has been done to estimate various parameters like strain, stress and strain energy density...
June 14, 2017: Scientific Reports
Heng Zhang, Nicolas Menguy, Fuxian Wang, Karim Benzerara, Eric Leroy, Peiyu Liu, Wenqi Liu, Chunli Wang, Yongxin Pan, Zhibao Chen, Jinhua Li
Magnetotactic bacteria (MTB) are morphologically and phylogenetically diverse prokaryotes. They can form intracellular chain-assembled magnetite (Fe3O4) or greigite (Fe3S4) nanocrystals each enveloped by a lipid bilayer membrane called a magnetosome. Magnetotactic cocci have been found to be the most abundant morphotypes of MTB in various aquatic environments. However, knowledge on magnetosome biomineralization within magnetotactic cocci remains elusive due to small number of strains that have been cultured...
2017: Frontiers in Microbiology
Antoine Hinaut, Baran Eren, Roland Steiner, Sara Freund, Res Jöhr, Thilo Glatzel, Laurent Marot, Ernst Meyer, Shigeki Kawai
Templating insulating surfaces at the nanoscale is an interesting prospect for applications that involve the adsorption of molecules or nanoparticles where electronic decoupling of the adsorbed species from the substrate is needed. In this study, we present a method to structure alkali halide surfaces at the nanoscale using a combination of low temperature plasma exposure and annealing, and characterize the surfaces by atomic force microscopy. We find that nanostructurating can be controlled by the duration of the exposure, the atomic mass of the plasma gas and the subsequent step-by-step annealing process...
June 21, 2017: Physical Chemistry Chemical Physics: PCCP
Laura S van Bezouwen, Stefano Caffarri, Ravindra S Kale, Roman Kouřil, Andy-Mark W H Thunnissen, Gert T Oostergetel, Egbert J Boekema
Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosynthetic membranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvesting complexes (LHCs), which act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C2S2M2 supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single-particle analysis at an overall resolution of 5.3 Å...
June 12, 2017: Nature Plants
Qing Li, Zheng Wang, Jiamei Mo, Guoxia Zhang, Yirui Chen, Chuchu Huang
Imaging the size distribution of metal nanoparticles (NPs) in a tissue has important implications in terms of evaluating NP toxicity. Microscopy techniques used to image tissue NPs are limited by complicated sample preparation or poor resolution. In this study, we developed a laser ablation (LA) system coupled to single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) for quantitative imaging of gold (G)NPs in tissue samples. In this system, GNPs were ablated but did not disintegrate and integrate under optimised operation conditions, which were verified by characterising LA particles by scanning electron microscopy...
June 7, 2017: Scientific Reports
Janette B Myers, Vincent Zaegel, Steven J Coultrap, Adam P Miller, K Ulrich Bayer, Steve L Reichow
The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15-35 nm in diameter...
June 7, 2017: Nature Communications
Pu Qian, Elizabeth C Martin, Irene W Ng, C Neil Hunter
In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba...
June 3, 2017: Biochimica et Biophysica Acta
Marcus D Wilson, Alessandro Costa
The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward...
June 1, 2017: Acta Crystallographica. Section D, Structural Biology
S Rawson, M J McPhillie, R M Johnson, C W G Fishwick, S P Muench
Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice...
June 1, 2017: Acta Crystallographica. Section D, Structural Biology
Edward P Morris, Paula C A da Fonseca
With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein-ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein-ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites...
June 1, 2017: Acta Crystallographica. Section D, Structural Biology
Rafael Fernandez-Leiro, Sjors H W Scheres
The formal concept of a workflow to single-particle analysis of cryo-electron microscopy (cryo-EM) images in the RELION program is described. In this approach, the structure-determination process is considered as a graph, where intermediate results in the form of images or metadata are the vertices, and different functionalities of the program are the edges. The new implementation automatically logs all user actions, facilitates file management and disk cleaning, and allows convenient browsing of the history of a project...
June 1, 2017: Acta Crystallographica. Section D, Structural Biology
Erik P Carter, Elif Ç Seymour, Steven M Scherr, George G Daaboul, David S Freedman, M Selim Ünlü, John H Connor
This chapter describes an approach for the label-free imaging and quantification of intact Ebola virus (EBOV) and EBOV viruslike particles (VLPs) using a light microscopy technique. In this technique, individual virus particles are captured onto a silicon chip that has been printed with spots of virus-specific capture antibodies. These captured virions are then detected using an optical approach called interference reflectance imaging. This approach allows for the detection of each virus particle that is captured on an antibody spot and can resolve the filamentous structure of EBOV VLPs without the need for electron microscopy...
2017: Methods in Molecular Biology
Dušan Turk
Macromolecular crystallography and electron microscopy (single-particle and in situ tomography) are merging into a single approach used by the two coalescing scientific communities. The merger is a consequence of technical developments that enabled determination of atomic structures of macromolecules by electron microscopy. Technological progress in experimental methods of macromolecular structure determination, computer hardware, and software changed and continues to change the nature of model building and visualization of molecular structures...
2017: Methods in Molecular Biology
Niyun Zhou, Hongwei Wang, Jiawei Wang
The resolution of electron-potential maps in single-particle cryo-electron microscopy (cryoEM) is approaching atomic or near- atomic resolution. However, no program currently exists for de novo cryoEM model building at resolutions exceeding beyond 3.5 Å. Here, we present a program, EMBuilder, based on template matching, to generate cryoEM models at high resolution. The program identifies features in both secondary-structure and Cα stages. In the secondary structure stage, helices and strands are identified with pre-computed templates, and the voxel size of the entire map is then refined to account for microscopic magnification errors...
June 1, 2017: Scientific Reports
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