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Cryo electron microscopy

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https://www.readbyqxmd.com/read/28547763/new-approaches-towards-the-understanding-of-integral-membrane-proteins-a-structural-perspective-on-g-protein-coupled-receptors
#1
REVIEW
Reinhard Grisshammer
Three-dimensional structure determination of integral membrane proteins has advanced in unprecedented detail our understanding of mechanistic events of how ion channels, transporters, receptors and enzymes function. This exciting progress required a tremendous amount of methods development, as exemplified here with G protein-coupled receptors (GPCRs): Optimizing the production of GPCRs in recombinant hosts; increasing the probability of crystal formation using high-affinity ligands, nanobodies, and minimal G proteins for co-crystallization, thus stabilizing receptors into one conformation; using the T4 lysozyme technology and other fusion partners to promote crystal contacts; advancing crystallization methods including the development of novel detergents, and miniaturization and automation of the lipidic cubic phase crystallization method; the concept of conformational thermostabilization of GPCRs; and developing microfocus X-ray synchrotron technologies to analyze small GPCR crystals...
May 25, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28546914/transmission-electron-microscopy-as-a-tool-for-the-characterization-of-soft-materials-application-and-interpretation
#2
REVIEW
Linda E Franken, Egbert J Boekema, Marc C A Stuart
Transmission electron microscopy (TEM) provides direct structural information on nano-structured materials and is popular as a characterization tool in soft matter and supramolecular chemistry. However, technical aspects of sample preparation are overlooked and erroneous image interpretations are regularly encountered in the literature. There are three most commonly used TEM methods as we derived from literature: drying, staining and cryo-TEM, which are explained here with respect to their application, limitations and interpretation...
May 2017: Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
https://www.readbyqxmd.com/read/28543996/self-assembly-of-perylenediimide-single-strand-dna-conjugates-employing-hydrophobic-interactions-and-dna-base-pairing-to-create-a-diverse-structural-space
#3
Frederick Lewis, Ashutosh Kumar Mishra, Haim Weissman, Elisha Krieg, Kevin A Voltaw, Martin McCullagh, Boris Rybtchiinski
The self-assembly behavior of DNA conjugates possessing a perylenediimide (PDI) head group and a single N-oligonucleotide tail has been investigated using a combination of optical spectroscopy and cryogenic transmission electron microscopy (cryo-TEM) imaging. In order to obtain insight into the interplay between DNA base pairing and PDI hydrophobic interactions we employed systematic variation in the length and composition of the oligo tails. Conjugates with short (TA)n or (CG)n oligo tails (n ≤ 3) form fibers (helical or nonhelical) constructed from -stacked PDI head groups with pendent oligo tails at room temperature in aqueous solution...
May 24, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/28543856/experimental-charge-density-from-electron-microscopic-maps
#4
Jimin Wang
The charge density (CD) distribution of an atom is the difference per unit volume between the positive charge of its nucleus and the distribution of the negative charges carried by the electrons that are associated with it. The CDs of the atoms in macromolecules are responsible for their electrostatic potential (ESP) distributions, which can now be visualized using cryo-electron microscopy at high resolution. CD maps can be recovered from experimental ESP density maps using the negative Laplacian operation...
May 20, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28541401/three-dimensional-structural-labeling-microscopy-of-cilia-and-flagella
#5
Toshiyuki Oda
Locating a molecule within a cell using protein-tagging and immunofluorescence is a fundamental technique in cell biology, whereas in three-dimensional electron microscopy, locating a subunit within a macromolecular complex remains challenging. Recently, we developed a new structural labeling method for cryo-electron tomography by taking advantage of the biotin-streptavidin system, and have intensively used this method to locate a number of proteins and protein domains in cilia and flagella. In this review, we summarize our findings on the three-dimensional architecture of the axoneme, especially the importance of coiled-coil proteins...
May 25, 2017: Microscopy
https://www.readbyqxmd.com/read/28538729/cryo-em-structure-of-the-activated-glp-1-receptor-in-complex-with-a-g-protein
#6
Yan Zhang, Bingfa Sun, Dan Feng, Hongli Hu, Matthew Chu, Qianhui Qu, Jeffrey T Tarrasch, Shane Li, Tong Sun Kobilka, Brian K Kobilka, Georgios Skiniotis
Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near atomic resolution...
May 24, 2017: Nature
https://www.readbyqxmd.com/read/28536302/a-cryo-em-based-model-of-phosphorylation-and-fkbp12-6-mediated-allosterism-of-the-cardiac-ryanodine-receptor
#7
Sonali Dhindwal, Joshua Lobo, Vanessa Cabra, Demetrio J Santiago, Ashok R Nayak, Kelly Dryden, Montserrat Samsó
Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain...
May 23, 2017: Science Signaling
https://www.readbyqxmd.com/read/28534487/rules-of-engagement-between-%C3%AE-v%C3%AE-6-integrin-and-foot-and-mouth-disease-virus
#8
Abhay Kotecha, Quan Wang, Xianchi Dong, Serban L Ilca, Marina Ondiviela, Rao Zihe, Julian Seago, Bryan Charleston, Elizabeth E Fry, Nicola G A Abrescia, Timothy A Springer, Juha T Huiskonen, David I Stuart
Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding...
May 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28533751/docetaxel-loaded-nanoparticles-assembled-from-%C3%AE-cyclodextrin-calixarene-giant-surfactants-physicochemical-properties-and-cytotoxic-effect-in-prostate-cancer-and-glioblastoma-cells
#9
Laura Gallego-Yerga, Inmaculada Posadas, Cristina de la Torre, Jesús Ruiz-Almansa, Francesco Sansone, Carmen Ortiz Mellet, Alessandro Casnati, José M García Fernández, Valentín Ceña
Giant amphiphiles encompassing a hydrophilic β-cyclodextrin (βCD) component and a hydrophobic calix[4]arene (CA4) module undergo self-assembly in aqueous media to afford core-shell nanospheres or nanocapsules, depending on the nanoprecipitation protocol, with high docetaxel (DTX) loading capacity. The blank and loaded nanoparticles have been fully characterized by dynamic light scattering (DLS), ζ-potential measurements and cryo-transmission electron microscopy (cryo-TEM). The data are compatible with the distribution of the drug between the nanoparticle core and the shell, where it is probably anchored by inclusion of the DTX aromatic moieties in βCD cavities...
2017: Frontiers in Pharmacology
https://www.readbyqxmd.com/read/28532216/structural-insights-into-the-eukaryotic-transcription-initiation-machinery
#10
Eva Nogales, Robert K Louder, Yuan He
Eukaryotic gene transcription requires the assembly at the promoter of a large preinitiation complex (PIC) that includes RNA polymerase II (Pol II) and the general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. The size and complexity of Pol II, TFIID, and TFIIH have precluded their reconstitution from heterologous systems, and purification relies on scarce endogenous sources. Together with their conformational flexibility and the transient nature of their interactions, these limitations had precluded structural characterization of the PIC...
May 22, 2017: Annual Review of Biophysics
https://www.readbyqxmd.com/read/28530806/photoluminescent-hybrids-of-cellulose-nanocrystals-and-carbon-quantum-dots-as-cytocompatible-probes-for-in-vitro-bio-imaging
#11
Jiaqi Guo, Dongfei Liu, Ilari Filpponen, Leena-Sisko Johansson, Jani-Markus Malho, Sakeena Quraishi, Falk Liebner, Hélder A Santos, Orlando J Rojas
We present an approach to construct biocompatible and photoluminescent hybrid materials comprised of carbon quantum dots (CQD) and TEMPO-oxidized cellulose nanocrystals (TO-CNC). First, the amino-functionalized carbon quantum dots (NH2-CQD) were synthesized using a simple microwave method and the TO-CNCs were prepared by hydrochloric acid (HCl) hydrolysis followed by TEMPO-mediated oxidation. The conjugation of NH2-CQD and TO-CNC was conducted via carbodiimide-assisted coupling chemistry. The synthesized TO-CNC@CQD hybrid nanomaterials were characterized using X-ray photoelectron spectroscopy, cryo-transmittance electron microscopy, confocal microscopy and fluorescence spectroscopy...
May 22, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28530653/structure-of-a-pre-catalytic-spliceosome
#12
Clemens Plaschka, Pei-Chun Lin, Kiyoshi Nagai
Intron removal requires assembly of the spliceosome on pre-mRNA and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 snRNP associates with U4/U6.U5 tri-snRNP through U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The U2 snRNP 3' region is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5'-splice site...
May 22, 2017: Nature
https://www.readbyqxmd.com/read/28528643/a-new-method-for-cryo-sectioning-cell-monolayers-using-a-correlative-workflow
#13
Androniki Kolovou, Martin Schorb, Abul Tarafder, Carsten Sachse, Yannick Schwab, Rachel Santarella-Mellwig
Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528638/triclem-combining-high-precision-room-temperature-clem-with%C3%A2-cryo-fluorescence-microscopy-to-identify-very-rare-events
#14
Nicholas R Ader, Wanda Kukulski
Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28524878/structure-of-the-quaternary-complex-between-srp-sr-and-translocon-bound-to-the-translating-ribosome
#15
Ahmad Jomaa, Yu-Hsien Hwang Fu, Daniel Boehringer, Marc Leibundgut, Shu-Ou Shan, Nenad Ban
During co-translational protein targeting, the signal recognition particle (SRP) binds to the translating ribosome displaying the signal sequence to deliver it to the SRP receptor (SR) on the membrane, where the signal peptide is transferred to the translocon. Using electron cryo-microscopy, we have determined the structure of a quaternary complex of the translating Escherichia coli ribosome, the SRP-SR in the 'activated' state and the translocon. Our structure, supported by biochemical experiments, reveals that the SRP RNA adopts a kinked and untwisted conformation to allow repositioning of the 'activated' SRP-SR complex on the ribosome...
May 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28522842/cryo-electron-microscopy-structure-of-the-macrobrachium-rosenbergii-nodavirus-capsid-at-7-angstroms-resolution
#16
Kok Lian Ho, Chare Li Kueh, Poay Ling Beh, Wen Siang Tan, David Bhella
White tail disease in the giant freshwater prawn Macrobrachium rosenbergii causes significant economic losses in shrimp farms and hatcheries and poses a threat to food-security in many developing countries. Outbreaks of Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease (WTD) are associated with up to 100% mortality rates. There are no interventions available to treat or prevent MrNV disease however. Here we show the structure of MrNV virus-like particles (VLPs) produced by recombinant expression of the capsid protein, using cryogenic electron microscopy...
May 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28514858/a-cholesterol-based-surface-active-ionic-liquid-that-can-form-microemulsions-and-spontaneous-vesicles
#17
Arghajit Pyne, Jagannath Kuchlyan, Chiranjit Maiti, Dibakar Dhara, Nilmoni Sarkar
In this article, we have reported the synthesis and physicochemical characterization of a novel L-glycine amino acid derived cholesterol based surface active ionic liquid (SAIL). This SAIL has been explored for the preparation of ionic liquid (IL)-in-oil microemulsions and vesicles. The formation of IL-in-oil microemulsion is characterized by construction of a ternary phase diagram, dynamic light scattering (DLS) measurement, proton nuclear magnetic resonance (1H NMR) study, fluorescence measurement using coumarin 480 (C-480) as a molecular probe and also by recording the diffusion behavior of the molecular probe rhodamine 6G (R6G) in microemulsion droplets through Fluorescence correlation spectroscopy (FCS) technique...
May 17, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
https://www.readbyqxmd.com/read/28513636/modular-synthesis-of-self-assembling-janus-dendrimers-and-facile-preparation-of-drug-loaded-dendrimersomes
#18
Sami Nummelin, Markus Selin, Sacha Legrand, Jarmo Ropponen, Jani Seitsonen, Antti Nykänen, Jari Koivisto, Jouni Hirvonen, Mauri A Kostiainen, Luis M Bimbo
Materials and methods aimed at the next generation of nanoscale carriers for drugs and other therapeutics are currently in great demand. Yet, creating these precise molecular arrangements in a feasible and straightforward manner represents a remarkable challenge. Herein we report a modular synthetic route for amphiphilic Janus-dendrimers via a copper-catalyzed click reaction (CuAAC) and a facile procedure, using simple injection, to obtain highly uniform dendrimersomes with efficient loading of the model drug compound propranolol...
May 17, 2017: Nanoscale
https://www.readbyqxmd.com/read/28510023/protein-rna-interactions-structural-biology-and-computational-modeling-techniques
#19
REVIEW
Susan Jones
RNA-binding proteins are functionally diverse within cells, being involved in RNA-metabolism, translation, DNA damage repair, and gene regulation at both the transcriptional and post-transcriptional levels. Much has been learnt about their interactions with RNAs through structure determination techniques and computational modeling. This review gives an overview of the structural data currently available for protein-RNA complexes, and discusses the technical issues facing structural biologists working to solve their structures...
December 2016: Biophysical Reviews
https://www.readbyqxmd.com/read/28510021/molecular-assembly-and-structure-of-the-bacteriophage-t4-tail
#20
REVIEW
Fumio Arisaka, Moh Lan Yap, Shuji Kanamaru, Michael G Rossmann
The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3...
December 2016: Biophysical Reviews
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