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Michael A Cianfrocco, Indrajit Lahiri, Frank DiMaio, Andres E Leschziner
Access to streamlined computational resources remains a significant bottleneck for new users of cryo-electron microscopy (cryo-EM). To address this, we have developed tools that will submit cryo-EM analysis routines and atomic model building jobs directly to Amazon Web Services (AWS) from a local computer or laptop. These new software tools ("cryoem-cloud-tools") have incorporated optimal data movement, security, and cost-saving strategies, giving novice users access to complex cryo-EM data processing pipelines...
June 1, 2018: Journal of Structural Biology
Alex J Noble, Venkata P Dandey, Hui Wei, Julia Brasch, Jillian Chase, Priyamvada Acharya, Yong Zi Tan, Zhening Zhang, Laura Y Kim, Giovanna Scapin, Micah Rapp, Edward T Eng, William J Rice, Anchi Cheng, Carl J Negro, Lawrence Shapiro, Peter D Kwong, David Jeruzalmi, Amedee des Georges, Clinton S Potter, Bridget Carragher
Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface...
May 29, 2018: ELife
Caleigh M Azumaya, Francisco Sierra-Valdez, Julio F Cordero-Morales, Terunaga Nakagawa
The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating...
May 11, 2018: Journal of Biological Chemistry
Xin Liang, Jonathon Howard
The sensations of sound, touch and pressure are mediated by mechanotransduction channels - transmembrane proteins whose ionic permeabilities are gated by mechanical forces. New structures of Piezo1 by cryoEM lead to the suggestion that this channel might sense membrane tension through changes in the local curvature of the membrane.
April 23, 2018: Current Biology: CB
Jenna K Rimel, Dylan J Taatjes
TFIIH is a 10-subunit complex that regulates RNA polymerase II (pol II) transcription but also serves other important biological roles. Although much remains unknown about TFIIH function in eukaryotic cells, much progress has been made even in just the past few years, due in part to technological advances (e.g. cryoEM and single molecule methods) and the development of chemical inhibitors of TFIIH enzymes. This review focuses on the major cellular roles for TFIIH, with an emphasis on TFIIH function as a regulator of pol II transcription...
June 2018: Protein Science: a Publication of the Protein Society
Xueni Li, Shiheng Liu, Jiansen Jiang, Lingdi Zhang, Sara Espinosa, Ryan C Hill, Kirk C Hansen, Z Hong Zhou, Rui Zhao
The originally published version of this Article contained several errors in Figure 2, panel a: the basepair register in SL3-4 of yeast U1 snRNA was depicted incorrectly; the basepair for A287-U295 in yeast U1 snRNA was erroneously present; basepairs for U84-G119, G309-U532, A288-U295 and U289-A294 in yeast U1 snRNA were missing; the bulging nucleotide in SL3 of human U1 snRNA was depicted as G instead of C; and the dashed boxes defining the 5' ss binding site and Sm site in both human and yeast snRNAs were not drawn accurately...
April 11, 2018: Nature Communications
Jinlan Gao, Hao Wang, Qipeng Yuan, Yue Feng
Photosynthesis converts solar energy into chemical energy to sustain all life on earth by providing oxygen and food, and controlling the atmospheric carbon dioxide. During this process, the water-splitting and oxygen-evolving reaction is catalyzed by photosystem II (PSII), while photosystem I (PSI) generates the reducing power for the reduction of NADP+ to NADPH. Together with their peripheral light-harvesting complexes (LHCs), photosystems function as multisubunit supercomplexes located in the thylakoid membranes of cyanobacteria, algae, and plants...
2018: Frontiers in Plant Science
Zhening Zhang, Wenguang G Liang, Lucas J Bailey, Yong Zi Tan, Hui Wei, Andrew Wang, Mara Farcasanu, Virgil A Woods, Lauren A McCord, David Lee, Weifeng Shang, Rebecca Deprez-Poulain, Benoit Deprez, David R Liu, Akiko Koide, Shohei Koide, Anthony A Kossiakoff, Sheng Li, Bridget Carragher, Clinton S Potter, Wei-Jen Tang
Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity...
March 29, 2018: ELife
Andrii Ishchenko, Cornelius Gati, Vadim Cherezov
G protein-coupled receptors mediate cell signaling and regulate the majority of sensory and physiological processes in the human body. Recent breakthroughs in cryo-electron microscopy and X-ray free electron lasers have accelerated structural studies of difficult-to-crystallize receptors and their signaling complexes, and have opened up new opportunities in understanding conformational dynamics and visualizing the process of receptor activation with unprecedented spatial and temporal resolution. Here, we summarize major milestones and challenges associated with the application of these techniques and outline future directions in their development with a focus on membrane protein structural biology...
March 16, 2018: Current Opinion in Structural Biology
Roman I Koning, Abraham J Koster, Thomas H Sharp
Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware...
May 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
Soung-Hun Roh, Nicholas J Stam, Corey F Hryc, Sergio Couoh-Cardel, Grigore Pintilie, Wah Chiu, Stephan Wilkens
The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+ -ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport...
March 15, 2018: Molecular Cell
Giovanna Scapin, Venkata P Dandey, Zhening Zhang, Winifred Prosise, Alan Hruza, Theresa Kelly, Todd Mayhood, Corey Strickland, Clinton S Potter, Bridget Carragher
The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain...
April 5, 2018: Nature
Kevin W Huynh, Jiansen Jiang, Natalia Abuladze, Kirill Tsirulnikov, Liyo Kao, Xuesi Shao, Debra Newman, Rustam Azimov, Alexander Pushkin, Z Hong Zhou, Ira Kurtz
Na+ -coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+ -coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3...
March 2, 2018: Nature Communications
Van Son Nguyen, Badreddine Douzi, Eric Durand, Alain Roussel, Eric Cascales, Christian Cambillau
The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM...
April 2018: Current Opinion in Structural Biology
Christopher J Russo, Richard Henderson
The fluctuating granularity or "bee swarm" effect seen in highly defocussed transmission electron micrographs is caused by microscopic charge fluctuations in the specimen created by the illuminating beam. In the field of high-resolution single particle electron cryomicroscopy (cryoEM), there has been a concern that this fluctuating charge might cause defocus-dependent Thon ring fading which would degrade the final image. In this paper, we have analysed the 2.35 Å fringes from the (111) reflection in images of gold nanoparticles embedded in amorphous ice...
April 2018: Ultramicroscopy
Christopher J Russo, Richard Henderson
When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work...
April 2018: Ultramicroscopy
Fuxing Zeng, Hong Jin
The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control...
February 5, 2018: Scientific Reports
Venkata P Dandey, Hui Wei, Zhening Zhang, Yong Zi Tan, Priyamvada Acharya, Edward T Eng, William J Rice, Peter A Kahn, Clinton S Potter, Bridget Carragher
We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.
May 2018: Journal of Structural Biology
Hui Wei, Venkata P Dandey, Zhening Zhang, Ashleigh Raczkowski, Willam J Rice, Bridget Carragher, Clinton S Potter
We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes...
May 2018: Journal of Structural Biology
Pascal Krotee, Sarah L Griner, Michael R Sawaya, Duilio Cascio, Jose A Rodriguez, Dan Shi, Stephan Philipp, Kevin Murray, Lorena Saelices, Ji Lee, Paul Seidler, Charles G Glabe, Lin Jiang, Tamir Gonen, David S Eisenberg
Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity...
February 23, 2018: Journal of Biological Chemistry
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