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Cryo-EM

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https://www.readbyqxmd.com/read/29036409/structure-of-an-innexin-gap-junction-channel-and-cryo-em-sample-preparation
#1
Atsunori Oshima
Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy...
September 21, 2017: Microscopy
https://www.readbyqxmd.com/read/29035579/beta-barrel-detection-for-medium-resolution-cryo-electron-microscopy-density-maps-using-genetic-algorithms-and-ray-tracing
#2
Albert Ng, Dong Si
Cryo-electron microscopy (cryo-EM) is a technique that produces three-dimensional density maps of large protein complexes. This allows for the study of the structure of these proteins. Identifying the secondary structures within proteins is vital to understanding the overall structure and function of the protein. The [Formula: see text]-barrel is one such secondary structure, commonly found in lipocalins and membrane proteins. In this article, we present a novel approach that utilizes genetic algorithms, kd-trees, and ray tracing to automatically detect and extract [Formula: see text]-barrels from cryo-EM density maps...
October 16, 2017: Journal of Computational Biology: a Journal of Computational Molecular Cell Biology
https://www.readbyqxmd.com/read/29035201/anti-diabetic-drug-binding-site-in-a-mammalian-katp-channels-revealed-by-cryo-em
#3
Gregory M Martin, Balamurugan Kandasamy, Frank DiMaio, Craig Yoshioka, Show-Ling Shyng
Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites...
October 16, 2017: ELife
https://www.readbyqxmd.com/read/29027901/structure-of-rna-polymerase-bound-to-ribosomal-30s-subunit
#4
Gabriel Demo, Aviram Rasouly, Nikita Vasilyev, Vladimir Svetlov, Anna B Loveland, Ruben Diaz-Avalos, Nikolaus Grigorieff, Evgeny Nudler, Andrei A Korostelev
In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit...
October 13, 2017: ELife
https://www.readbyqxmd.com/read/29021523/high-fidelity-dna-replication-in-mycobacterium-tuberculosis-relies-on-a-trinuclear-zinc-center
#5
Soledad Baños-Mateos, Anne-Marie M van Roon, Ulla F Lang, Sarah L Maslen, J Mark Skehel, Meindert H Lamers
High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues...
October 11, 2017: Nature Communications
https://www.readbyqxmd.com/read/29021343/tubulin-isoform-composition-tunes-microtubule-dynamics
#6
Annapurna Vemu, Joseph Atherton, Jeffrey O Spector, Carolyn A Moores, Antonina Roll-Mecak
Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4...
October 11, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29019979/cryo-electron-microscopy-structure-of-the-lysosomal-calcium-permeable-channel-trpml3
#7
Marscha Hirschi, Mark A Herzik, Jinhong Wie, Yang Suo, William F Borschel, Dejian Ren, Gabriel C Lander, Seok-Yong Lee
The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca(2+)-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca(2+) from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype...
October 11, 2017: Nature
https://www.readbyqxmd.com/read/28993151/using-a-smalp-platform-to-determine-a-sub-nm-single-particle-cryo-em-membrane-protein-structure
#8
Mayuriben Parmar, Shaun Rawson, Charlotte A Scarff, Adrian Goldman, Timothy R Dafforn, Stephen P Muench, Vincent L G Postis
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs...
October 6, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28991891/achieving-better-than-3-%C3%A3-resolution-by-single-particle-cryo-em-at-200-kev
#9
Mark A Herzik, Mengyu Wu, Gabriel C Lander
Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
October 9, 2017: Nature Methods
https://www.readbyqxmd.com/read/28989723/single-particle-cryo-em-using-alignment-by-classification-abc-the-structure-of-lumbricus-terrestris-haemoglobin
#10
Pavel Afanasyev, Charlotte Seer-Linnemayr, Raimond B G Ravelli, Rishi Matadeen, Sacha De Carlo, Bart Alewijnse, Rodrigo V Portugal, Navraj S Pannu, Michael Schatz, Marin van Heel
Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets...
September 1, 2017: IUCrJ
https://www.readbyqxmd.com/read/28985564/cryo-em-structures-reveal-mechanism-and-inhibition-of-dna-targeting-by-a-crispr-cas-surveillance-complex
#11
Tai Wei Guo, Alberto Bartesaghi, Hui Yang, Veronica Falconieri, Prashant Rao, Alan Merk, Edward T Eng, Ashleigh M Raczkowski, Tara Fox, Lesley A Earl, Dinshaw J Patel, Sriram Subramaniam
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved...
October 5, 2017: Cell
https://www.readbyqxmd.com/read/28981077/cryo-electron-microscopy-snapshots-of-the-spliceosome-structural-insights-into-a-dynamic-ribonucleoprotein-machine
#12
REVIEW
Sebastian M Fica, Kiyoshi Nagai
The spliceosome excises introns from pre-messenger RNAs using an RNA-based active site that is cradled by a dynamic protein scaffold. A recent revolution in cryo-electron microscopy (cryo-EM) has led to near-atomic-resolution structures of key spliceosome complexes that provide insight into the mechanism of activation, splice site positioning, catalysis, protein rearrangements and ATPase-mediated dynamics of the active site. The cryo-EM structures rationalize decades of observations from genetic and biochemical studies and provide a molecular framework for future functional studies...
October 5, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28981073/clarifying-the-supercomplex-the-higher-order-organization-of-the-mitochondrial-electron-transport-chain
#13
REVIEW
James A Letts, Leonid A Sazanov
The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI-CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII2 and CIV (SC I+III2+IV, known as the respirasome), as well as with CIII2 alone (SC I+III2)...
October 5, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28975896/length-of-encapsidated-cargo-impacts-stability-and-structure-of-in-vitro-assembled-alphavirus-core-like-particles
#14
Vamseedhar Rayaprolu, Alan Moore, Joseph Che-Yen Wang, Boon Chong Goh, Juan Perilla, Adam Zlotnick, Suchetana Mukhopadhyay
In vitro assembly of alphavirus nucleocapsid cores, called core-like particles (CLPs), require a polyanionic cargo. There are no sequence or structure requirements to encapsidate single-stranded nucleic acid cargo. In this work, we wanted to determine how the length of the cargo impacts the stability and structure of the assembled CLPs. We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein and, if the cargo is long enough, will also act to scaffold the CP monomers together...
October 4, 2017: Journal of Physics. Condensed Matter: An Institute of Physics Journal
https://www.readbyqxmd.com/read/28971542/scipion-web-tools-easy-to-use-cryo-em-image-processing-over-the-web
#15
Pablo Conesa Mingo, José Gutierrez, Adrián Quintana, José Miguel de la Rosa Trevín, Airén Zaldívar-Peraza, Jesús Cuenca Alba, Moshen Kazemi, Javier Vargas, Laura Del Cano, Joan Segura, Carlos Oscar S Sorzano, Jose María Carazo
Macromolecular structural determination by Electron Microscopy under cryogenic conditions is revolutionizing the field of structural biology, interesting a large community of potential users. Still, the path from raw images to density maps is complex, and sophisticated image processing suites are required in this process, often demanding the installation and understanding of different software packages. Here we present Scipion Web Tools, a web-based set of tools/workflows derived from the Scipion image processing framework, specially tailored to non-expert users in need of very precise answers at several key stages of the structural elucidation process...
October 3, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28963369/tools-for-the-cryo-em-gold-rush-going-from-the-cryo-em-map-to-the-atomistic-model
#16
Doo Nam Kim, Karissa Sanbonmatsu
As cryo electron microscopy (Cryo-EM) enters mainstream structural biology, the demand for fitting methods is high. Here, we explain the importance of flexible fitting for Cryo-EM, potential concerns and assessment methods of this approach, and review existing methods. We aim to give readers concrete descriptions of Cryo-EM flexible fitting methods with corresponding examples.
September 29, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28961740/emhp-an-accurate-automated-hole-masking-algorithm-for-single-particle-cryo-em-image-processing
#17
Zachary Berndsen, Charles Bowman, Haerin Jang, Andrew B Ward
Summary: The Electron Microscopy Hole Punch (EMHP) is a streamlined suite of tools for quick assessment, sorting, and hole masking of electron micrographs. With recent advances in single-particle electron cryo-microscopy (cryo-EM) data processing allowing for the rapid determination of protein structures using a smaller computational footprint, we saw the need for a fast and simple tool for data pre-processing that could run independent of existing high-performance computing (HPC) infrastructures...
August 7, 2017: Bioinformatics
https://www.readbyqxmd.com/read/28959035/the-cryo-em-structure-of-hibernating-100s-ribosome-dimer-from-pathogenic-staphylococcus-aureus
#18
Donna Matzov, Shintaro Aibara, Arnab Basu, Ella Zimmerman, Anat Bashan, Mee-Ngan F Yap, Alexey Amunts, Ada E Yonath
Formation of 100S ribosome dimer is generally associated with translation suppression in bacteria. Trans-acting factors ribosome modulation factor (RMF) and hibernating promoting factor (HPF) were shown to directly mediate this process in E. coli. Gram-positive S. aureus lacks an RMF homolog and the structural basis for its 100S formation was not known. Here we report the cryo-electron microscopy structure of the native 100S ribosome from S. aureus, revealing the molecular mechanism of its formation. The structure is distinct from previously reported analogs and relies on the HPF C-terminal extension forming the binding platform for the interactions between both of the small ribosomal subunits...
September 28, 2017: Nature Communications
https://www.readbyqxmd.com/read/28951391/structure-and-rna-recognition-of-ribosome-assembly-factor-utp30
#19
Jianfei Hu, Xing Zhu, Keqiong Ye
The 90S pre-ribosomes are gigantic early assembly intermediates of small ribosomal subunits. Cryo-EM structures of 90S were recently determined, but many of its components have not been accurately modelled. Here we determine the crystal structure of yeast Utp30, a ribosomal L1 domain-containing protein in 90S, at 2.65 Å resolution, revealing a classic two-domain fold. The structure of Utp30 fits well into the cryo-EM density of 90S, confirming its previously assigned location. Utp30 binds to the rearranged helix 41 of 18S rRNA and helix 4 of 5' external transcribed spacer in 90S...
September 26, 2017: RNA
https://www.readbyqxmd.com/read/28950085/structural-biology-a-force-sensitive-ion-channel-springs-to-life
#20
Xin Liang, Jonathan Howard
The sensations of sound, acceleration and touch are mediated by mechanotransduction channels, which convert mechanical stimuli into electrical responses. The structure of one such channel, NOMPC, was recently solved by cryo-EM, revealing a bundle of helices that may act as coiled springs to transmit the forces that open the channel.
September 25, 2017: Current Biology: CB
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