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cryo EM

Yueyong Xin, Yang Shi, Tongxin Niu, Qingqiang Wang, Wanqiang Niu, Xiaojun Huang, Wei Ding, Lei Yang, Robert E Blankenship, Xiaoling Xu, Fei Sun
Photosynthetic prokaryotes evolved diverse light-harvesting (LH) antennas to absorb sunlight and transfer energy to reaction centers (RC). The filamentous anoxygenic phototrophs (FAPs) are important early branching photosynthetic bacteria in understanding the origin and evolution of photosynthesis. How their photosynthetic machinery assembles for efficient energy transfer is yet to be elucidated. Here, we report the 4.1 Å structure of photosynthetic core complex from Roseiflexus castenholzii by cryo-electron microscopy...
April 19, 2018: Nature Communications
Dongjie Zhu, Xiangxi Wang, Qianglin Fang, James L Van Etten, Michael G Rossmann, Zihe Rao, Xinzheng Zhang
The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a "block-based" reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes...
April 19, 2018: Nature Communications
Alain Dautant, Thomas Meier, Alexander Hahn, Déborah Tribouillard-Tanvier, Jean-Paul di Rago, Roza Kucharczyk
Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae , we can begin to understand these molecular machines and their associated defects at the molecular level...
2018: Frontiers in Physiology
Fabrizio Martino, Mohinder Pal, Hugo Muñoz-Hernández, Carlos F Rodríguez, Rafael Núñez-Ramírez, David Gil-Carton, Gianluca Degliesposti, J Mark Skehel, S Mark Roe, Chrisostomos Prodromou, Laurence H Pearl, Oscar Llorca
The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90...
April 16, 2018: Nature Communications
Saori Maki-Yonekura, Rei Matsuoka, Yoshiki Yamashita, Hirofumi Shimizu, Maiko Tanaka, Fumie Iwabuki, Koji Yonekura
Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM...
April 17, 2018: ELife
Michael C Regan, Timothy Grant, Miranda J McDaniel, Erkan Karakas, Jing Zhang, Stephen F Traynelis, Nikolaus Grigorieff, Hiro Furukawa
Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the exon-5-encoded motif at the amino-terminal domain (ATD) of the GluN1 subunit are known to display robustly altered deactivation rates and pH sensitivity, but the underlying mechanism for this functional modification is largely unknown. Here, we show through cryoelectron microscopy (cryo-EM) that the presence of the exon 5 motif in GluN1 alters the local architecture of heterotetrameric GluN1-GluN2 NMDA receptors and creates contacts with the ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, which are absent in NMDA receptors lacking the exon 5 motif...
April 10, 2018: Neuron
Lesley A Earl, Veronica Falconieri, Sriram Subramaniam
Over the past few years, the advances in technology and methods that have revolutionized cryo-EM are allowing for key insights in a variety of areas in biology, and microbiology is no exception. A wide range of important macromolecular assemblies in prokaryotic and eukaryotic cells, as well as intact viruses, have now become accessible to investigation by new methods in 3D electron microscopy. We focus here on selected examples that illustrate this breadth, and review the application of methods in single particle cryo-EM and cryo-electron tomography to progress in the structural biology of CRISPR systems, visualization of small molecule drugs in membrane proteins, in situ visualization of bacterial nanomachines, and the analysis of antigen-antibody interactions to drive vaccine design...
April 12, 2018: Current Opinion in Microbiology
Anurag P Srivastava, Min Luo, Wenchang Zhou, Jindrich Symersky, Dongyang Bai, Melissa G Chambers, José D Faraldo-Gómez, Maofu Liao, David M Mueller
Mitochondrial ATP synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-EM to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin, at 3.6 Å and 3.8 Å resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ-subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10 -ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway and show how oligomycin blocks ATP synthesis...
April 12, 2018: Science
Rianne Bartelds, Mohammad Hadi Nematollahi, Tjeerd Pols, Marc C A Stuart, Abbas Pardakhty, Gholamreza Asadikaram, Bert Poolman
Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidylethanolamine lipids plus cholesterol. Niosomes are highly stable and only slightly more leaky than liposomes as assayed by calcein leakage; the permeability for ions (KCl) is higher than that of liposomes...
2018: PloS One
Min-Sung Kim, Watchalee Chuenchor, Xuemin Chen, Yanxiang Cui, Xing Zhang, Z Hong Zhou, Martin Gellert, Wei Yang
To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation...
March 29, 2018: Molecular Cell
Huping Wang, Wenyu Han, Junichi Takagi, Yao Cong
Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining (NS) or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" (YISPANL) strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes...
April 3, 2018: Journal of Molecular Biology
Mihai Ciubotaru, Mihaela Georgiana Musat, Marius Surleac, Elena Ionita, Andrei Jose Petrescu, Edgars Abele, Ramona Abele
Currently used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes, changes these viral enzymes which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations...
April 5, 2018: Current Medicinal Chemistry
Shuai Yuan, Jialing Wang, Dongjie Zhu, Nan Wang, Qiang Gao, Wenyuan Chen, Hao Tang, Junzhi Wang, Xinzheng Zhang, Hongrong Liu, Zihe Rao, Xiangxi Wang
Structurally and genetically, human herpesviruses are among the largest and most complex of viruses. Using cryo-electron microscopy (cryo-EM) with an optimized image reconstruction strategy, we report the herpes simplex virus type 2 (HSV-2) capsid structure at 3.1 angstroms, which is built up of about 3000 proteins organized into three types of hexons (central, peripentonal, and edge), pentons, and triplexes. Both hexons and pentons contain the major capsid protein, VP5; hexons also contain a small capsid protein, VP26; and triplexes comprise VP23 and VP19C...
April 6, 2018: Science
Pu Qian, C Alistair Siebert, Peiyi Wang, Daniel P Canniffe, C Neil Hunter
The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 β-polypeptides sandwiched between 17 α- and 16 γ-polypeptides...
April 4, 2018: Nature
Nian Zhang, Hui Chen, Yujiao Fan, Lu Zhou, Sylvain Trépout, Jia Guo, Min-Hui Li
Fluorescent polymersomes are interesting systems for cell/tissue imaging and in vivo study of drug distribution and delivery. We report on bright fluorescent polymersomes with aggregation-induced emission self-assembled by a series of tetraphenylethylene(TPE)-containing amphiphilic biodegradable block copolymers, where the hydrophilic block is a polyethyleneglycol PEG and hydrophobic block is a TPE-substituted trimethylenecarbonate polymer P(TPE-TMC). Their self-assemblies in water were prepared by nanoprecipitation using dioxane or THF as co-solvent, and the self-assembling processes were studied in detail by cryo-EM, DLS and spectrofluorometer...
April 4, 2018: ACS Nano
Scott M Jackson, Ioannis Manolaridis, Julia Kowal, Melanie Zechner, Nicholas M I Taylor, Manuel Bause, Stefanie Bauer, Ruben Bartholomaeus, Guenther Bernhardt, Burkhard Koenig, Armin Buschauer, Henning Stahlberg, Karl-Heinz Altmann, Kaspar P Locher
ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar...
April 2, 2018: Nature Structural & Molecular Biology
Piotr Neumann, Achim Dickmanns, Ralf Ficner
Recent advances in instrumentation and image-processing software have resulted in a resolution revolution in cryo-electron microscopy (cryo-EM) and a surge in the popularity of this technique. However, despite technical progress and hundreds of structures determined so far, development of standards assessing the agreement between the cryo-EM map and the respective model has fallen behind. Here we establish a validation procedure evaluating this agreement and applied it to a set of 565 cryo-EM structures. Analysis of the results revealed that three-quarters of the validated structures exhibit moderate or low agreement between the map and the corresponding model, mostly due to limited structural features possessed by these maps...
March 19, 2018: Structure
Wei Lin, Kalyan Das, David Degen, Abhishek Mazumder, Diego Duchi, Dongye Wang, Yon W Ebright, Richard Y Ebright, Elena Sineva, Matthew Gigliotti, Aashish Srivastava, Sukhendu Mandal, Yi Jiang, Yu Liu, Ruiheng Yin, Zhening Zhang, Edward T Eng, Dennis Thomas, Stefano Donadio, Haibo Zhang, Changsheng Zhang, Achillefs N Kapanidis, Richard H Ebright
Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state...
March 28, 2018: Molecular Cell
Sandip Kaledhonkar, Ziao Fu, Howard White, Joachim Frank
With the advent of direct electron detectors, cryo-EM has become a popular choice for molecular structure determination. Among its advantages over X-ray crystallography are (1) no need for crystals, (2) much smaller sample volumes, and (3) the ability to determine multiple structures or conformations coexisting in one sample. In principle, kinetic experiments can be done using standard cryo-EM, but mixing and freezing grids require several seconds. However, many biological processes are much faster than that time scale, and the ensuing short-lived states of the molecules cannot be captured...
2018: Methods in Molecular Biology
Jing-Xiang Wu, Dian Ding, Mengmeng Wang, Yunlu Kang, Xin Zeng, Lei Chen
ATP-sensitive potassium channels (KATP ) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic KATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic KATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6...
March 28, 2018: Protein & Cell
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