Unsupervised cryo-EM data clustering through adaptively constrained K-means algorithm

In single-particle cryo-electron microscopy (cryo-EM), K-means clustering algorithm is widely used in unsupervised 2D classification of projection images of biological macromolecules. 3D ab initio reconstruction requires accurate unsupervised classification in order to separate molecular projections of distinct orientations. Due to background noise in single-particle images and uncertainty of molecular orientations, traditional K-means clustering algorithm may classify images into wrong classes and produce classes with a large variation in membership. Overcoming these limitations requires further development on clustering algorithms for cryo-EM data analysis. We propose a novel unsupervised data clustering method building upon the traditional K-means algorithm. By introducing an adaptive constraint term in the objective function, our algorithm not only avoids a large variation in class sizes but also produces more accurate data clustering. Applications of this approach to both simulated and experimental cryo-EM data demonstrate that our algorithm is a significantly improved alterative to the traditional K-means algorithm in single-particle cryo-EM analysis.Comment: 35 pages, 14 figure

Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca2+-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 angstrom and a resolution of 4.2 angstrom for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca2+ activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.Strategic Priority Research Program of Chinese Academy of Sciences [XDB08030202]; National Basic Research Program (973 Program); Ministry of Science & Technology of China [2012CB917200, 2014CB910700]; National Natural Science Foundation of China [31270768]; Ministry of Education of China (111 Program China)SCI(E)PubMed中国科技核心期刊(ISTIC)[email protected]; [email protected]

Mechanisms for ·O2- and ·OH Production on Flowerlike BiVO4 Photocatalysis Based on Electron Spin Resonance

Many studies have focused on the use of BiVO4 as a photocatalyst, but few have investigated the production of free radicals during the photocatalytic process. Following synthesis of flowerlike BiVO4 and characterization by X-ray diffraction (XRD), Raman spectroscopy, Scanning electron microscopy (SEM) Scanning electron microscopy (EDX), UV-Vis and XPS, we successfully prepared BiVO4. Then we used electron spin resonance (ESR) to determine the production and degradation of individual active free radicals, including the superoxide radical (·O2-) and the hydroxyl radical (·OH). In the first experiment, we used ESR to detect the signals of free radicals (·O2- and ·OH) under varying oxygen conditions. The results shown that in addition to production by ·O2-, ·OH could also be produced by oxidation of h+ to OH−. In the next experiment, we detected ·OH under varying pH to identify the result of the first experiment, and found that signal intensities increased with increasing pH, indicating the mechanism for ·OH production. Finally, we conducted a trapping experiment to examine free radical degradation mechanisms. We identified ·OH and h+ as the main active free radicals and showed the complete production about ·OH. These results improve current knowledge of free radical production mechanisms, which can be used to enhance the photocatalytic performance of BiVO4

Unsupervised Cryo-EM Data Clustering through Adaptively Constrained K-Means Algorithm - Fig 3

<p><b>2D class averages of GroEL using the traditional K-means (a), EQK-means (b) and ACK-means (c) in MRA approach from SPARX</b>. Class size is shown at the left bottom of each class average. ACK-means (b) is the best by having the most number of clear classes.</p

Unsupervised Cryo-EM Data Clustering through Adaptively Constrained K-Means Algorithm - Fig 10

<p><b>2D class averages of RP using the traditional K-means (a) and ACK-means (b) in MRA/MSA from EMAN2.</b> Class size is shown at the left bottom of each class average. Classes generated by ACK-means (a) are clearer than those by the traditional K-means (a).</p

Comparison of classification results of simulated data with SNR = 1/10.

<p>The First column (panels a, c and e) is the normalized histogram of angular distances. More accurate classification produces curve with higher peak concentrated at lower angular distance. The second column (panels b, d and f) shows the class sizes arranged in an ascend order. The most balanced classification has a horizontal line in this plot. (a) and (b) are from experiments using different clustering algorithms in MRA approach under SPARX. (c) and (d) are from experiments using different clustering algorithms in MRA/MSA approach under EMAN2. (e) and (f) are from experiments using different clustering algorithms in RFA approach under SPIDER. In all graphs, red curves present the results from the ACK-means algorithm.</p

The running time of different algorithms in different approaches.

<p>The running time of different algorithms in different approaches.</p

Convergence of K-means, EQK-means and ACK-means in MRA.

<p>The three algorithms behave similarly as iteration increases, converging very fast at the first several iterations.</p

www.mdpi.com/journal/ijms Detection of Promyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARα) Fusion Gene with Functionalized Graphene Oxide

Abstract: An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable