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https://www.readbyqxmd.com/read/27914893/near-atomic-resolution-structure-determination-of-a-cypovirus-capsid-and-polymerase-complex-using-cryo-em-at-200kv
#1
Xiaowu Li, Niyun Zhou, Wenyuan Chen, Bin Zhu, Xurong Wang, Bin Xu, Jiawei Wang, Hongrong Liu, Lingpeng Cheng
Single particle cryo-electron microscopy (cryo-EM) allows high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5Å) cryo-EM structures reported to date were obtained by using 300kV transmission electron microscopes (TEMs). We report here the structures of a cypovirus capsid of 750Å diameter at 3.3Å resolution and RNA-dependent RNA polymerase (RdRp) complexes within the capsid at 3.9Å resolution using a 200kV TEM. The newly resolved structure revealed conformational changes of two subdomains in the RdRp...
November 30, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27912063/structure-of-mammalian-respiratory-supercomplex-i1iii2iv1
#2
Meng Wu, Jinke Gu, Runyu Guo, Yushen Huang, Maojun Yang
The mammalian respiratory chain complexes assemble into supercomplexes (SCs) and reside in the inner mitochondrial membrane to transfer electrons and establish the proton gradient for complex V to synthesize ATP. The precise arrangement of SCs is largely unknown. Here, we report a 4.0-Å cryo-electron microscopy (cryo-EM) structure of the major SC in porcine heart, the 1.7-MDa SCI1III2IV1. The complex III (CIII) dimer and complex IV (CIV) bind at the same side of the L-shaped complex I (CI). Several accessory or supernumerary subunits of CI, such as NDUFA11, NDUFB4, NDUFB8, and NDUFB9, directly contribute to the oligomerization of CI, CIII, and CIV...
December 1, 2016: Cell
https://www.readbyqxmd.com/read/27912054/inner-secrets-of-the-respirasome
#3
Andrew Melber, Dennis R Winge
Structure determination by cryo-electron microscopy has approached atomic resolution and helped solve structures of large membrane-protein complexes that resisted crystallography. The 4.0 Å cryo-EM structure of one of the most intricate enzyme systems, the respirasome, in the mitochondrial inner membrane is reported in this issue of Cell.
December 1, 2016: Cell
https://www.readbyqxmd.com/read/27909983/4-4-%C3%A3-resolution-cryo-em-structure-of-human-mtor-complex-1
#4
Huirong Yang, Jia Wang, Mengjie Liu, Xizi Chen, Min Huang, Dan Tan, Meng-Qiu Dong, Catherine C L Wong, Jiawei Wang, Yanhui Xu, Hong-Wei Wang
Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics...
December 1, 2016: Protein & Cell
https://www.readbyqxmd.com/read/27905396/atomic-structure-of-the-innexin-6-gap-junction-channel-determined-by-cryo-em
#5
Atsunori Oshima, Kazutoshi Tani, Yoshinori Fujiyoshi
Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity...
December 1, 2016: Nature Communications
https://www.readbyqxmd.com/read/27898298/estimation-of-visibility-of-phase-contrast-with-extraction-voltages-for-field-emission-gun-electron-microscopes
#6
Xing Meng
Estimation was made for visibility of phase contrast with varying extraction voltages. The resulting decay rates of visibility show that images with low image contrast from cryo EM will be seriously impacted with high extraction voltages.
November 19, 2016: Ultramicroscopy
https://www.readbyqxmd.com/read/27867008/molecular-structures-of-transcribing-rna-polymerase-i
#7
Lucas Tafur, Yashar Sadian, Niklas A Hoffmann, Arjen J Jakobi, Rene Wetzel, Wim J H Hagen, Carsten Sachse, Christoph W Müller
RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes...
November 16, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27864160/blotting-free-and-lossless-cryo-electron-microscopy-grid-preparation-from-nanoliter-sized-protein-samples-and-single-cell-extracts
#8
Stefan A Arnold, Stefan Albiez, Andrej Bieri, Anastasia Syntychaki, Ricardo Adaixo, Robb A McLeod, Kenneth N Goldie, Henning Stahlberg, Thomas Braun
We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3 to 20 nanoliters of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics...
November 15, 2016: Journal of Structural Biology
https://www.readbyqxmd.com/read/27863656/engineering-thermosensitive-liposome-nanoparticle-hybrids-loaded-with-doxorubicin-for-heat-triggered-drug-release
#9
Zahraa Al-Ahmady, Neus Lozano, Kuo-Ching Mei, Wafa' T Al-Jamal, Kostas Kostarelos
The engineering of responsive multifunctional delivery systems that combine therapeutic and diagnostic (theranostic) capabilities holds great promise and interest. We describe the design of thermosensitive liposome-nanoparticle (NP) hybrids that can modulate drug release in response to external heating stimulus. These hybrid systems were successfully engineered by the incorporation of gold, silver, and iron oxide NPs into the lipid bilayer of lysolipid-containing thermosensitive liposomes (LTSL). Structural characterization of LTSL-NP hybrids using cryo-EM and AFM revealed the incorporation of metallic NPs into the lipid membranes without compromising doxorubicin loading and retention capability...
November 30, 2016: International Journal of Pharmaceutics
https://www.readbyqxmd.com/read/27860084/cryo-em-as-a-tool-for-structure-based-drug-development
#10
Felipe Merino, Stefan Raunser
For decades, X-ray crystallography and NMR have been the major techniques to study the atomic structure of macromolecules. However, because of size, instability, low yield, and other factors, many macromolecules are recalcitrant to crystallization or NMR studies. Electron cryo microscopy (cryo-EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that could not be crystallized, but atomic resolution was mostly beyond its reach. Capable of sensing directly the incident electrons, a new generation of detectors has recently revolutionized the field, with structures of macromolecules being now solved routinely to near-atomic resolution...
November 10, 2016: Angewandte Chemie
https://www.readbyqxmd.com/read/27851956/structural-basis-for-the-activation-of-ikk1-%C3%AE
#11
Smarajit Polley, Dario Oliveira Passos, De-Bin Huang, Maria Carmen Mulero, Anup Mazumder, Tapan Biswas, Inder M Verma, Dmitry Lyumkis, Gourisankar Ghosh
Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers...
November 15, 2016: Cell Reports
https://www.readbyqxmd.com/read/27849524/building-proteins-in-a-day-efficient-3d-molecular-structure-estimation-with-electron-cryomicroscopy
#12
Ali Punjani, Marcus Brubaker, David Fleet
Discovering the 3D atomic-resolution structure of molecules such as proteins and viruses is one of the foremost research problems in biology and medicine. Electron Cryomicroscopy (cryo-EM) is a promising vision-based technique for structure estimation which attempts to reconstruct 3D atomic structures from a large set of 2D transmission electron microscope images. This paper presents a new Bayesian framework for cryo-EM structure estimation that builds on modern stochastic optimization techniques to allow one to scale to very large datasets...
November 10, 2016: IEEE Transactions on Pattern Analysis and Machine Intelligence
https://www.readbyqxmd.com/read/27845625/accelerated-cryo-em-structure-determination-with-parallelisation-using-gpus-in-relion-2
#13
Dari Kimanius, Björn O Forsberg, Sjors Hw Scheres, Erik Lindahl
By reaching near-atomic resolution for a wide range of specimens, single-particle cryo-EM structure determination is transforming structural biology. However, the necessary calculations come at increased computational costs, introducing a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the RELION image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure determination workflow...
November 15, 2016: ELife
https://www.readbyqxmd.com/read/27830641/functional-asymmetry-and-electron-flow-in-the-bovine-respirasome
#14
Joana S Sousa, Deryck J Mills, Janet Vonck, Werner Kühlbrandt
Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons...
November 10, 2016: ELife
https://www.readbyqxmd.com/read/27821763/design-of-a-molecular-support-for-cryo-em-structure-determination
#15
Thomas G Martin, Tanmay A M Bharat, Andreas C Joerger, Xiao-Chen Bai, Florian Praetorius, Alan R Fersht, Hendrik Dietz, Sjors H W Scheres
Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA...
November 22, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27821762/x-ray-and-cryo-em-structures-of-monomeric-and-filamentous-actin-like-protein-mamk-reveal-changes-associated-with-polymerization
#16
Jan Löwe, Shaoda He, Sjors H W Scheres, Christos G Savva
Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB...
November 22, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27821609/structure-and-mechanism-of-the-atp-synthase-membrane-motor-inferred-from-quantitative-integrative-modeling
#17
Vanessa Leone, José D Faraldo-Gómez
Two subunits within the transmembrane domain of the ATP synthase-the c-ring and subunit a-energize the production of 90% of cellular ATP by transducing an electrochemical gradient of H(+) or Na(+) into rotational motion. The nature of this turbine-like energy conversion mechanism has been elusive for decades, owing to the lack of definitive structural information on subunit a or its c-ring interface. In a recent breakthrough, several structures of this complex were resolved by cryo-electron microscopy (cryo-EM), but the modest resolution of the data has led to divergent interpretations...
December 2016: Journal of General Physiology
https://www.readbyqxmd.com/read/27819266/cryo-em-study-of-start-codon-selection-during-archaeal-translation-initiation
#18
Pierre-Damien Coureux, Christine Lazennec-Schurdevin, Auriane Monestier, Eric Larquet, Lionel Cladière, Bruno P Klaholz, Emmanuelle Schmitt, Yves Mechulam
Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAi(Met)) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site...
November 7, 2016: Nature Communications
https://www.readbyqxmd.com/read/27819028/symmetry-mismatch-reconstruction-of-genomes-and-associated-proteins-within-icosahedral-viruses-using-cryo-em
#19
Xiaowu Li, Hongrong Liu, Lingpeng Cheng
Although near-atomic resolutions have been routinely achieved for structural determination of many icosahedral viral capsids, structures of genomes and associated proteins within the capsids are still less characterized because the genome information is overlapped by the highly symmetric capsid information in the virus particle images. We recently developed a software package for symmetry-mismatch structural reconstruction and determined the structures of the genome and RNA polymerases within an icosahedral virus for the first time...
2016: Biophysics Reports
https://www.readbyqxmd.com/read/27818103/key-intermediates-in-ribosome-recycling-visualized-by-time-resolved-cryoelectron-microscopy
#20
Ziao Fu, Sandip Kaledhonkar, Anneli Borg, Ming Sun, Bo Chen, Robert A Grassucci, Måns Ehrenberg, Joachim Frank
Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner...
November 2, 2016: Structure
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