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Dramatic improvement of crystals of large RNAs by cation replacement and dehydration.

ABSTRACT: Compared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and counterions for RNA folding. Replacement of Li(+) and Mg(2+) needed for growth of crystals of a tRNA-riboswitch-protein complex with Sr(2+), coupled with dehydration, dramatically improved the resolution limit (8.5-3.2 Å) and data quality, enabling structure determination. The soft Sr(2+) ion forms numerous stabilizing intermolecular contacts. Comparison of pre- and posttreatment structures reveals how RNA assemblies redistribute as quasi-rigid bodies to yield improved crystal packing. Cation exchange complements previously reported postcrystallization dehydration of protein crystals and represents a potentially general strategy for improving crystals of large RNAs.

SUBMITTER: Zhang J

PROVIDER: S-EPMC4177851 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Dramatic improvement of crystals of large RNAs by cation replacement and dehydration.

Zhang Jinwei J Ferré-D'Amaré Adrian R AR

Structure (London, England : 1993) 20140901 9

Compared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and ...[more]

PMID: 25185828

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Project description:BACKGROUND:Regional analgesia is more effective than conventional analgesia for controlling pain and may facilitate rehabilitation after large joint replacement in the short term. It remains unclear if regional anaesthesia improves functional outcomes after joint replacement beyond three months after surgery. OBJECTIVES:To assess the effects of regional anaesthesia and analgesia on long-term functional outcomes 3, 6 and 12 months after elective major joint (knee, shoulder and hip) replacement surgery. SEARCH METHODS:We performed an electronic search of several databases (CENTRAL, MEDLINE, EMBASE, CINAHL), and handsearched reference lists and conference abstracts. We updated our search in June 2015. SELECTION CRITERIA:We included randomized controlled trials (RCTs) comparing regional analgesia versus conventional analgesia in patients undergoing total shoulder, hip or knee replacement. We included studies that reported a functional outcome with a follow-up of at least three months after surgery. DATA COLLECTION AND ANALYSIS:We used standard methodological procedures expected by Cochrane. We contacted study authors for additional information. MAIN RESULTS:We included six studies with 350 participants followed for at least three months. All of these studies enrolled participants undergoing total knee replacement. Studies were at least partially blinded. Three studies had a high risk of performance bias and one a high risk of attrition bias, but the risk of bias was otherwise unclear or low.Only one study assessed joint function using a global score. Due to heterogeneity in outcome and reporting, we could only pool three out of six RCTs, with range of motion assessed at three months after surgery used as a surrogate for joint function. All studies had a high risk of detection bias. Using the random-effects model, there was no statistically significant difference between the experimental and control groups (mean difference 3.99 degrees, 95% confidence interval (CI) - 2.23 to 10.21; P value = 0.21, 3 studies, 140 participants, very low quality evidence).We did not perform further analyses because immediate adverse effects were not part of the explicit outcomes of any of these typically small studies, and long-term adverse events after regional anaesthesia are rare.None of the included studies elicited or reported long-term adverse effects like persistent nerve damage. AUTHORS' CONCLUSIONS:More high-quality studies are needed to establish the effects of regional analgesia on function after major joint replacement, as well as on the risk of adverse events (falls).

Project description:BackgroundLong non-coding RNAs (lncRNAs) play important roles in plant growth and stress responses. Studies of lncRNAs in non-model plants are quite limited, especially those investigating multiple dehydration stresses. In this study, we identified novel lncRNAs and analyzed their functions in dehydration stress memory in switchgrass, an excellent biofuel feedstock and soil-conserving plant in the Gramineae family.ResultsWe analyzed genome-wide transcriptional profiles of leaves of 5-week-old switchgrass plantlets grown via tissue culture after primary and secondary dehydration stresses (D1 and D2) and identified 16,551 novel lncRNAs, including 4554 annotated lncRNAs (targeting 3574 genes), and 11,997 unknown lncRNAs. Gene ontology and pathway enrichment analysis of annotated genes showed that the differentially expressed lncRNAs were related to abscisic acid (ABA) and ethylene (ETH) biosynthesis and signal transduction, and to starch and sucrose metabolism. The upregulated lncRNAs and genes were related to ABA synthesis and its signal transduction, and to trehalose synthesis. Meanwhile, lncRNAs and genes related to ETH biosynthesis and signal transduction were suppressed. LncRNAs and genes involved in ABA metabolism were verified using quantitative real-time PCR, and the endogenous ABA content was determined via high performance liquid chromatography mass spectrometry (HPLC-MS). These results showed that ABA accumulated significantly during dehydration stress, especially in D2. Furthermore, we identified 307 dehydration stress memory lncRNAs, and the ratios of different memory types in switchgrass were similar to those in Arabidopsis and maize.ConclusionsThe molecular responses of switchgrass lncRNAs to multiple dehydration stresses were researched systematically, revealing novel information about their transcriptional regulatory behavior. This study provides new insights into the response mechanism to dehydration stress in plants. The lncRNAs and pathways identified in this study provide valuable information for genetic modification of switchgrass and other crops.

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Dramatic improvement of crystals of large RNAs by cation replacement and dehydration.

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Dramatic improvement of crystals of large RNAs by cation replacement and dehydration.

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