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Table 6 High likelihood targets for miR-132 determined by minimum free energy (dG) values representing structural accessibility of miR-132 binding sites on the target gene.
Structures were solved by molecular replacement using PHASER (McCoy et al., 2005 ) and were refined against maximum likelihood targets using REFMAC (Murshudov et al., 1997 ).
The model was refined with PHENIX.refine (Adams et al., 2010), using gradient minimization with weight optimization, maximum likelihood targets, non-crystallographic symmetry constraints, individual B-factors, and TLS parameters.
Further simulated annealing refinement with torsion angle molecular dynamics in CNS followed by 10 cycles of restrained refinement on maximum likelihood targets gave an R-factor of 0.425 and R-free of 0.469.
Structures were solved by molecular replacement using PHASER (McCoy et al., 2007) and were refined against maximum likelihood targets using restrained refinement and TLS protocols implemented in REFMAC (Murshudov et al., 1997).
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Using a multilevel, integrated coherent bioinformatics workflow led to the nomination of three high likelihood target genes for miR-132 with possible relevance for learned safety: the already experimentally validated target gene MeCP252 and the predicted, potential target genes GAT1 and PTEN.
Remark 3. Within our framework, a crude non-Bayesian single frame maximum likelihood target detector could be built by simply evaluating the likelihood map for each aspect state and finding the maximum over the image grid of the sum of likelihood maps weighted by the a priori probability for each state (usually assumed to be identical).
The structure was refined in Crystallography and NMR System (CNS) by using standard simulated annealing protocols and the amplitude-based maximum likelihood target function [38] [39].
The TargetScan5.0 algorithm identified the zinc finger E-box binding homeobox 2 (Zeb2/SIP1/ZFXH1B) gene as the highest likelihood target gene of the miR-200 family with 6 potential miR-429/miR-200b/miR-200c binding sites and an additional 3 potential miR-141/miR-200a binding sites in its 3'UTR.
The subsequent refinement consisted of alternating rounds of positional minimization, individual restrained thermal factor refinement, both using the MLF maximum likelihood target function, and model building.
The model was refined using gradient minimization with weight optimization and maximum-likelihood targets, TLS refinement, and individual atomic B-factor refinement.
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