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Secondary structures of RNAs are crucial to the understanding of their tertiary structures and functions. At present, many theoretical methods are widely used to predict RNA secondary structures. The performance of these methods has been evaluated but only for their ability of base-pairing prediction. However, the topology of a RNA secondary structure is more important for understanding its tertiary structure and function, especially for long RNAs. In this paper, we constructed a new non-redundant RNA database containing 73 RNA with lengths of 50-300 nucleotides and benchmarked four popular algorithms for both base pairing and topology. The results show that the prediction accuracy of secondary structure topology is only 38%, in contrast to 70% for that of base pairing. Furthermore, the topological consistency is not strongly correlated to the base-pairing consistency. Our results will be helpful to understand the limitations of RNA secondary structure prediction methods from a different point of view and also to their improvements in future.
Electron spin resonance imaging (ESRI) has been developed to detect the spatial contribution of free radicals in recent years, but all of these studies are used in animal systems and almost of them using exogenous spin probes and only a few to study the endogenous free radicals in vivo. However, there is no report about the endogenous free radical three-dimensional (3D) EPRI in plant because of low concentration of endogenous free radicals and low resolution of the L-band ESRI. Recently, we have developed the imaging resolution better than 200 lm which is higher 10 times than L-band (about 1-3 mm) and the sensitivity is higher about 1000 times than that of L-band ESRI. Using this system and spin trapping technique, we studied spatial contribution of the endogenous nitric oxide (NO) radical generation in wheat leaves and got a clear 3D ESRI picture and CT (computed tomography) of NO of a wheat leaf. This imaging picture shows a clear spatial distribution of NO free radicals in the leaf. This is the first 3D ESRI of endogenous NO free radical generated in plant in the world. We have also studied the distributions of NO generation in different plants from different species with different shapes and it is shown that this is a convenient method to study the endogenous free radicals in plants.
Correlative light and electron microscopy is a powerful technique for identification and determination of the structures of interested macromolecules in situ. Combined with sample vitrification, it would be much easier to preserve the native state of macromolecule complexes and distinguish them from the crowded structure environment. In this article, we present a detailed process for the application of the CorrSight system, a light microscope equipped with a cryo module, in combination with a cryo-electron microscope. A relatively long course of up to 7-8 h for cryo module preparation and multichannel light microscopy imaging of vitrified specimen can be sustained. Correlation of light and electron microscopy images at both grid levels to locate squares and square level to locate target particles, and verification of target particles can be performed with the help of AutoEMation software. Cryo-electron tomography is used for obtaining the three-dimensional structure information.
ATP-binding cassette (ABC) transporters are found in all forms of life from microbes to humans, and transport a wide variety of substrates across the cell membrane using the energy released from ATP hydrolysis and an alternating-access mechanism. MsbA is a homodimeric ABC exporter from Gramnegative bacteria, and transports amphipathic substrates including precursors of lipopolysaccharides from the inner leaflet to the outer leaflet of the cytoplasmic membrane. Despite extensive structural and functional studies, controversies remain regarding the dynamic properties of the conformational changes of MsbA during its transport cycle in the lipid environment. Here, we used single-molecule fluorescence resonance energy transfer (smFRET) to explore the dynamic behaviors of MsbA in detergent micelles, nanodiscs, and proteoliposomes. MsbA reconstituted into liposomes showed higher transition frequency between different states on the cytoplasmic side, whereas detergent-solubilized MsbA showed higher transition frequency on the periplasmic side. Three major states were identified from this smFRET study in the functional cycle of MsbA, including an intermediate conformation between the fully opened and fully closed cytoplasmic conformations, associated with both ATP binding and hydrolysis.