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Cryo electron microscopy

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https://www.readbyqxmd.com/read/28437792/phase-plate-cryo-em-structure-of-a-class-b-gpcr-g-protein-complex
#1
Yi-Lynn Liang, Maryam Khoshouei, Mazdak Radjainia, Yan Zhang, Alisa Glukhova, Jeffrey Tarrasch, David M Thal, Sebastian G B Furness, George Christopoulos, Thomas Coudrat, Radostin Danev, Wolfgang Baumeister, Laurence J Miller, Arthur Christopoulos, Brian K Kobilka, Denise Wootten, Georgios Skiniotis, Patrick M Sexton
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsβγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7...
April 24, 2017: Nature
https://www.readbyqxmd.com/read/28434999/smfret-experiments-of-the-rna-polymerase-ii-transcription-initiation-complex
#2
REVIEW
Nicole Malkusch, Thilo Dörfler, Julia Nagy, Tobias Eilert, Jens Michaelis
Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET(1)) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28431243/cryo-em-structure-of-the-open-human-ether-%C3%A3-go-go-related-k-channel-herg
#3
Weiwei Wang, Roderick MacKinnon
The human ether-à-go-go-related potassium channel (hERG, Kv11.1) is a voltage-dependent channel known for its role in repolarizing the cardiac action potential. hERG alteration by mutation or pharmacological inhibition produces Long QT syndrome and the lethal cardiac arrhythmia torsade de pointes. We have determined the molecular structure of hERG to 3.8 Å using cryo-electron microscopy. In this structure, the voltage sensors adopt a depolarized conformation, and the pore is open. The central cavity has an atypically small central volume surrounded by four deep hydrophobic pockets, which may explain hERG's unusual sensitivity to many drugs...
April 20, 2017: Cell
https://www.readbyqxmd.com/read/28428974/perspective-a-toolbox-for-protein-structure-determination-in-physiological-environment-through-oriented-2d-ordered-site-specific-immobilization
#4
M Altissimo, M Kiskinova, R Mincigrucci, L Vaccari, C Guarnaccia, C Masciovecchio
Revealing the structure of complex biological macromolecules, such as proteins, is an essential step for understanding the chemical mechanisms that determine the diversity of their functions. Synchrotron based X-ray crystallography and cryo-electron microscopy have made major contributions in determining thousands of protein structures even from micro-sized crystals. They suffer from some limitations that have not been overcome, such as radiation damage, the natural inability to crystallize a number of proteins, and experimental conditions for structure determination that are incompatible with the physiological environment...
July 2017: Structural Dynamics (Melville, N.Y.)
https://www.readbyqxmd.com/read/28424519/mechanism-of-chromatin-remodelling-revealed-by-the-snf2-nucleosome-structure
#5
Xiaoyu Liu, Meijing Li, Xian Xia, Xueming Li, Zhucheng Chen
Chromatin remodellers are helicase-like, ATP-dependent enzymes that alter chromatin structure and nucleosome positions to allow regulatory proteins access to DNA. Here we report the cryo-electron microscopy structure of chromatin remodeller Switch/sucrose non-fermentable (SWI2/SNF2) from Saccharomyces cerevisiae bound to the nucleosome. The structure shows that the two core domains of Snf2 are realigned upon nucleosome binding, suggesting activation of the enzyme. The core domains contact each other through two induced Brace helices, which are crucial for coupling ATP hydrolysis to chromatin remodelling...
April 19, 2017: Nature
https://www.readbyqxmd.com/read/28423342/a-broadly-neutralizing-antibody-targets-the-dynamic-hiv-envelope-trimer-apex-via-a-long-rigidified-and-anionic-%C3%AE-hairpin-structure
#6
Jeong Hyun Lee, Raiees Andrabi, Ching-Yao Su, Anila Yasmeen, Jean-Philippe Julien, Leopold Kong, Nicholas C Wu, Ryan McBride, Devin Sok, Matthias Pauthner, Christopher A Cottrell, Travis Nieusma, Claudia Blattner, James C Paulson, Per Johan Klasse, Ian A Wilson, Dennis R Burton, Andrew B Ward
Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature...
April 18, 2017: Immunity
https://www.readbyqxmd.com/read/28419915/enhanced-imaging-of-lipid-rich-nanoparticles-embedded-in-methylcellulose-films-for-transmission-electron-microscopy-using-mixtures-of-heavy-metals
#7
Jalal Asadi, Sophie Ferguson, Hussain Raja, Christian Hacker, Phedra Marius, Richard Ward, Christos Pliotas, James Naismith, John Lucocq
Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability...
April 10, 2017: Micron: the International Research and Review Journal for Microscopy
https://www.readbyqxmd.com/read/28416636/p53-dynamically-directs-tfiid-assembly-on-target-gene-promoters
#8
R A Coleman, Z Qiao, S K Singh, C S Peng, M Cianfrocco, Z Zhang, A Piasecka, H Aldeborgh, G Basishvili, W L Liu
p53 is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry...
April 17, 2017: Molecular and Cellular Biology
https://www.readbyqxmd.com/read/28412289/structural-studies-of-the-endogenous-spliceosome-the-supraspliceosome
#9
REVIEW
Joseph Sperling, Ruth Sperling
Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21 MDa) and highly dynamic molecular machine - the supraspliceosome - that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA...
April 12, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28409456/cryo-immuno-electron-microscopy-of-peroxisomal-marker-proteins
#10
Karina Mildner, Dagmar Zeuschner
Electron microscopy samples processed for cryo-immunogold-labeling need to be gently fixed to keep their antigenicity. Biological material like cultured cells or tissue can be prepared according to the standard Tokuyasu fixation or in a further developed rehydration method based on high-pressure freezing. We will describe here the variant and common steps of both methods in detail and illustrate their potency in the ultrastructural imaging of peroxisomes.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28409029/biophysical-approaches-facilitate-computational-drug-discovery-for-atp-binding-cassette-proteins
#11
REVIEW
Steven V Molinski, Zoltán Bozóky, Surtaj H Iram, Saumel Ahmadi
Although membrane proteins represent most therapeutically relevant drug targets, the availability of atomic resolution structures for this class of proteins has been limited. Structural characterization has been hampered by the biophysical nature of these polytopic transporters, receptors, and channels, and recent innovations to in vitro techniques aim to mitigate these challenges. One such class of membrane proteins, the ATP-binding cassette (ABC) superfamily, are broadly expressed throughout the human body, required for normal physiology and disease-causing when mutated, yet lacks sufficient structural representation in the Protein Data Bank...
2017: International Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/28408394/a-close-up-look-at-the-spliceosome-at-last
#12
John Abelson
Major developments in cryo-electron microscopy in the past three or four years have led to the solution of a number of spliceosome structures at high resolution, e.g., the fully assembled but not yet active spliceosome (Bact), the spliceosome just after the first step of splicing (C), and the spliceosome activated for the second step (C*). Therefore 30 years of genetics and biochemistry of the spliceosome can now be interpreted at the structural level. I have closely examined the RNase H domain of Prp8 in each of the structures...
April 13, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28393837/cryo-em-structures-of-mers-cov-and-sars-cov-spike-glycoproteins-reveal-the-dynamic-receptor-binding-domains
#13
Yuan Yuan, Duanfang Cao, Yanfang Zhang, Jun Ma, Jianxun Qi, Qihui Wang, Guangwen Lu, Ying Wu, Jinghua Yan, Yi Shi, Xinzheng Zhang, George F Gao
The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD)...
April 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28393831/determining-the-bacterial-cell-biology-of-planctomycetes
#14
Christian Boedeker, Margarete Schüler, Greta Reintjes, Olga Jeske, Muriel C F van Teeseling, Mareike Jogler, Patrick Rast, Daniela Borchert, Damien P Devos, Martin Kucklick, Miroslava Schaffer, Roberto Kolter, Laura van Niftrik, Susanne Engelmann, Rudolf Amann, Manfred Rohde, Harald Engelhardt, Christian Jogler
Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail...
April 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28392566/assembly-structure-function-and-regulation-of-type-iii-secretion-systems
#15
REVIEW
Wanyin Deng, Natalie C Marshall, Jennifer L Rowland, James M McCoy, Liam J Worrall, Andrew S Santos, Natalie C J Strynadka, B Brett Finlay
Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs...
April 10, 2017: Nature Reviews. Microbiology
https://www.readbyqxmd.com/read/28391678/investigation-of-novel-triple-responsive-wormlike-micelles
#16
Mingwei Zhao, Mingwei Gao, Caili Dai, Chenwei Zou, Zhe Yang, Xuepeng Wu, Yifei Liu, Yining Wu, Sisi Fang, Wenjiao Lv
Smart wormlike micelles with stimuli-tunable rheological properties may be useful in a variety of applications, such as in molecular devices and sensors. The formation of triplestimuli-responsive systems so far has been a challenging and important issue. In this work, a novel triplestimuli (photo-, pH-, and thermoresponsive) wormlike micelle is constructed with N-cetyl-N-methylmorpholinium bromide and trans-cinnamic acid (CA). The corresponding multiresponsive behaviors of wormlike micellar system were revealed using cryogenic transmission electron microscopy, a rheometer, and (1)H NMR...
April 20, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
https://www.readbyqxmd.com/read/28382301/bayesian-modeling-of-biomolecular-assemblies-with-cryo-em-maps
#17
Michael Habeck
A growing array of experimental techniques allows us to characterize the three-dimensional structure of large biological assemblies at increasingly higher resolution. In addition to X-ray crystallography and nuclear magnetic resonance in solution, new structure determination methods such cryo-electron microscopy (cryo-EM), crosslinking/mass spectrometry and solid-state NMR have emerged. Often it is not sufficient to use a single experimental method, but complementary data need to be collected by using multiple techniques...
2017: Frontiers in Molecular Biosciences
https://www.readbyqxmd.com/read/28380371/the-enigma-of-the-respiratory-chain-supercomplex
#18
REVIEW
Dusanka Milenkovic, James N Blaza, Nils-Göran Larsson, Judy Hirst
Respiratory chain dysfunction plays an important role in human disease and aging. It is now well established that the individual respiratory complexes can be organized into supercomplexes, and structures for these macromolecular assemblies, determined by electron cryo-microscopy, have been described recently. Nevertheless, the reason why supercomplexes exist remains an enigma. The widely held view that they enhance catalysis by channeling substrates is challenged by both structural and biophysical information...
April 4, 2017: Cell Metabolism
https://www.readbyqxmd.com/read/28377272/interactions-and-release-of-two-palmitoyl-peptides-from-phytantriol-cubosomes
#19
Seyedeh Parinaz Akhlaghi, Watson Loh
Phytantriol cubosomes loaded with two palmitoyl peptides (Palpepcubes), namely GHKcube and GQPRcube, were prepared using an ultrasonication protocol. The Palpepcubes dimensions were characterized by dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). Small-angle X-ray scattering (SAXS) analyses revealed that the bicontinuous cubic structure remained even at palmitoyl peptide contents as high as 5 wt.%, with an increase in the cell parameter from approximately 6.5 to 7.2 nm. Isothermal titration calorimetry (ITC) was used to elucidate the interactions between the blank cubosomes and the palmitoyl peptides, revealing an exothermic process of interaction...
April 1, 2017: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/28373698/the-structures-of-a-naturally-empty-cowpea-mosaic-virus-particle-and-its-genome-containing-counterpart-by-cryo-electron-microscopy
#20
Emma L Hesketh, Yulia Meshcheriakova, Rebecca F Thompson, George P Lomonossoff, Neil A Ranson
Cowpea mosaic virus (CPMV) is a picorna-like plant virus. As well as an intrinsic interest in CPMV as a plant pathogen, CPMV is of major interest in biotechnology applications such as nanotechnology. Here, we report high resolution cryo electron microscopy (cryo-EM) maps of wild type CPMV containing RNA-2, and of naturally-formed empty CPMV capsids. The resolution of these structures is sufficient to visualise large amino acids. We have refined an atomic model for each map and identified an essential amino acid involved in genome encapsidation...
April 3, 2017: Scientific Reports
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