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Cryo-EM

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https://www.readbyqxmd.com/read/28819229/cryo-em-analysis-of-homodimeric-full-length-lrrk2-and-lrrk1-protein-complexes
#1
Kushal Sejwal, Mohamed Chami, Hervé Rémigy, Renée Vancraenenbroeck, William Sibran, Rosmarie Sütterlin, Paul Baumgartner, Robert McLeod, Marie-Christine Chartier-Harlin, Veerle Baekelandt, Henning Stahlberg, Jean-Marc Taymans
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson's disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images...
August 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28813641/aim2-inflammasome-activation-and-regulation-a-structural-perspective
#2
Bing Wang, Qian Yin
Absent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic dsDNA and induces cytokine maturation, release and pyroptosis. It is composed of AIM2, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Recent X-ray crystallographic and high resolution cryo-electron microscopic (cryo-EM) studies have revealed a series of structures in AIM2 inflammasome activation and regulation. One prominent feature common in multiple steps is the assembly of high-order structures, especially helical filaments nucleated by upstream molecules, rather than stoichiometric complexes...
August 13, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/28809395/cryo-em-structures-of-the-80s-ribosomes-from-human-parasites-trichomonas-vaginalis-and-toxoplasma-gondii
#3
Zhifei Li, Qiang Guo, Lvqin Zheng, Yongsheng Ji, Yi-Ting Xie, De-Hua Lai, Zhao-Rong Lun, Xun Suo, Ning Gao
As an indispensable molecular machine universal in all living organisms, the ribosome has been selected by evolution to be the natural target of many antibiotics and small-molecule inhibitors. High-resolution structures of pathogen ribosomes are crucial for understanding the general and unique aspects of translation control in disease-causing microbes. With cryo-electron microscopy technique, we have determined structures of the cytosolic ribosomes from two human parasites, Trichomonas vaginalis and Toxoplasma gondii, at resolution of 3...
August 15, 2017: Cell Research
https://www.readbyqxmd.com/read/28802041/cryo-em-structure-of-the-tom-core-complex-from-neurospora-crassa
#4
Thomas Bausewein, Deryck J Mills, Julian D Langer, Beate Nitschke, Stephan Nussberger, Werner Kühlbrandt
The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7...
August 10, 2017: Cell
https://www.readbyqxmd.com/read/28795512/single-particle-cryo-em-improved-ab-initio-3d-reconstruction-with-simple-prime
#5
Cyril F Reboul, Michael Eager, Dominika Elmlund, Hans Elmlund
Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here we extend our previous work on PRIME and introduce new stochastic optimisation algorithms that improve the robustness of the approach...
August 10, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28783150/katanin-spiral-and-ring-structures-shed-light-on-power-stroke-for-microtubule-severing
#6
Elena Zehr, Agnieszka Szyk, Grzegorz Piszczek, Ewa Szczesna, Xiaobing Zuo, Antonina Roll-Mecak
Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function...
August 7, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28781996/opinion-hazards-faced-by-macromolecules-when-confined-to-thin-aqueous-films
#7
REVIEW
Robert M Glaeser, Bong-Gyoon Han
Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air-water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air-water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions...
2017: Biophysics Reports
https://www.readbyqxmd.com/read/28781166/cryo-em-structure-of-a-pre-catalytic-human-spliceosome-primed-for-activation
#8
Karl Bertram, Dmitry E Agafonov, Olexandr Dybkov, David Haselbach, Majety N Leelaram, Cindy L Will, Henning Urlaub, Berthold Kastner, Reinhard Lührmann, Holger Stark
Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step...
August 10, 2017: Cell
https://www.readbyqxmd.com/read/28779844/saposin-lipoprotein-scaffolds-for-structure-determination-of-membrane-transporters
#9
Joseph A Lyons, Andreas Bøggild, Poul Nissen, Jens Frauenfeld
Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin-lipid-protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28779836/lipid-nanodiscs-as-a-tool-for-high-resolution-structure-determination-of-membrane-proteins-by-single-particle-cryo-em
#10
Rouslan G Efremov, Christos Gatsogiannis, Stefan Raunser
The "resolution revolution" in electron cryomicroscopy (cryo-EM) profoundly changed structural biology of membrane proteins. Near-atomic structures of medium size to large membrane protein complexes can now be determined without crystallization. This significantly accelerates structure determination and also the visualization of small bound ligands. There is an additional advantage: the structure of membrane proteins can now be studied in their native or nearly native lipid bilayer environment. A popular lipid bilayer mimetic are lipid nanodiscs, which have been thoroughly characterized and successfully utilized in multiple applications...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28774558/molecular-dynamics-of-the-cryo-em-cftr-structure
#11
Hedvig Tordai, Ibolya Leveles, Tamás Hegedűs
Cystic fibrosis (CF), a lethal monogenic disease, is caused by mutant variants of the CF transmembrane conductance regulator (CFTR). Recent advances in single molecule cryo-EM methods enabled structural determination of full-length human and zebrafish CFTR, achieving an important milestone for CF drug development. To relate these structures to the gating cycle, we examined its dynamic features using molecular dynamics simulations. Our results show that the nucleotide binding domains (NBDs) in this bottom-open apo conformation exhibit motions related to dimerization and the bottom-closed apo CFTR model indicates opening of NBDs in contrast to transporters...
July 31, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28767037/cryo-em-structure-of-the-saga-and-nua4-coactivator-subunit-tra1-at-3-7-angstrom-resolution
#12
Luis Miguel Diaz-Santin, Natasha Lukoyanova, Emir Aciyan, Alan Cm Cheung
Coactivator complexes SAGA and NuA4 stimulate transcription by post-translationally modifying chromatin. Both complexes contain the Tra1 subunit, a highly conserved 3744-residue protein from the Phosphoinositide 3-Kinase-related kinase (PIKK) family and a direct target for multiple sequence-specific activators. We present the Cryo-EM structure of Saccharomyces cerevsisae Tra1 to 3.7 Å resolution, revealing an extensive network of alpha-helical solenoids organized into a diamond ring conformation and is strikingly reminiscent of DNA-PKcs, suggesting a direct role for Tra1 in DNA repair...
August 2, 2017: ELife
https://www.readbyqxmd.com/read/28759049/structural-basis-of-tir-domain-assembly-formation-in-mal-and-myd88-dependent-tlr4-signaling
#13
Thomas Ve, Parimala R Vajjhala, Andrew Hedger, Tristan Croll, Frank DiMaio, Shane Horsefield, Xiong Yu, Peter Lavrencic, Zahid Hassan, Garry P Morgan, Ashley Mansell, Mehdi Mobli, Ailis O'Carroll, Brieuc Chauvin, Yann Gambin, Emma Sierecki, Michael J Landsberg, Katryn J Stacey, Edward H Egelman, Bostjan Kobe
Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement...
July 31, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28757251/the-molecular-architecture-for-rna-guided-rna-cleavage-by-cas13a
#14
Liang Liu, Xueyan Li, Jun Ma, Zongqiang Li, Lilan You, Jiuyu Wang, Min Wang, Xinzheng Zhang, Yanli Wang
Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding...
August 10, 2017: Cell
https://www.readbyqxmd.com/read/28757144/refined-cryo-em-structure-of-the-t4-tail-tube-exploring-the-lowest-dose-limit
#15
Weili Zheng, Fengbin Wang, Nicholas M I Taylor, Ricardo C Guerrero-Ferreira, Petr G Leiman, Edward H Egelman
The bacteriophage T4 contractile tail (containing a tube and sheath) was the first biological assembly reconstructed in three dimensions by electron microscopy at a resolution of ∼35 Å in 1968. A single-particle reconstruction of the T4 baseplate was able to generate a 4.1 Å resolution map for the first two rings of the tube using the overall baseplate for alignment. We have now reconstructed the T4 tail tube at a resolution of 3.4 Å, more than a 1,000-fold increase in information content for the tube from 1968...
July 10, 2017: Structure
https://www.readbyqxmd.com/read/28756276/cryo-electron-microscopy-for-structural-analysis-of-dynamic-biological-macromolecules
#16
REVIEW
Kazuyoshi Murata, Matthias Wolf
BACKGROUND: Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules. SCOPE OF REVIEW: We provide an overview of the cryo-EM method and introduce contemporary studies to investigate biomolecular structure and dynamics, including examples from the recent literature...
July 27, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28750325/cryo-em-maps-reveal-five-fold-channel-structures-and-their-modification-by-gatekeeper-mutations-in-the-parvovirus-minute-virus-of-mice-mvm-capsid
#17
Suriyasri Subramanian, Lindsey J Organtini, Alec Grossman, Phillip P Domeier, Javier O Cifuente, Alexander M Makhov, James F Conway, Anthony D'Abramo, Susan F Cotmore, Peter Tattersall, Susan Hafenstein
In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively...
July 24, 2017: Virology
https://www.readbyqxmd.com/read/28747707/the-cryo-em-structure-of-gastric-h-k-atpase-with-bound-byk99-a-high-affinity-member-of-k-competitive-imidazo-1-2-a-pyridine-inhibitors
#18
Kazuhiro Abe, Jun Shimokawa, Mao Naito, Keith Munson, Olga Vagin, George Sachs, Hiroshi Suzuki, Kazutoshi Tani, Yoshinori Fujiyoshi
The gastric proton pump H(+),K(+)-ATPase acidifies the gastric lumen, and thus its inhibitors, including the imidazo[1,2-a]pyridine class of K(+)-competitive acid blockers (P-CABs), have potential application as acid-suppressing drugs. We determined the electron crystallographic structure of H(+),K(+)-ATPase at 6.5 Å resolution in the E2P state with bound BYK99, a potent P-CAB with a restricted ring structure. The BYK99 bound structure has an almost identical profile to that of a previously determined structure with bound SCH28080, the original P-CAB prototype, but is significantly different from the previously reported P-CAB-free form, illustrating a common conformational change is required for P-CAB binding...
July 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28741611/an-antimicrobial-peptide-that-inhibits-translation-by-trapping-release-factors-on-the-ribosome
#19
Tanja Florin, Cristina Maracci, Michael Graf, Prajwal Karki, Dorota Klepacki, Otto Berninghausen, Roland Beckmann, Nora Vázquez-Laslop, Daniel N Wilson, Marina V Rodnina, Alexander S Mankin
Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain...
July 24, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28739580/structural-insights-into-transcription-initiation-by-yeast-rna-polymerase-i
#20
Yashar Sadian, Lucas Tafur, Jan Kosinski, Arjen J Jakobi, Rene Wetzel, Katarzyna Buczak, Wim Jh Hagen, Martin Beck, Carsten Sachse, Christoph W Müller
In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold...
July 24, 2017: EMBO Journal
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