18 research outputs found
The different predictive performance of the MFS method, the SeqonlyMFS method, the Evolutionary Trace server, and the ConSurf server on two examples.
<p>The structure of an ornithine decarboxylase (A) (PDB identifier 1ord-A) and a cellobiohydrolase (B) (PDB identifier 1cel-A) are shown in the ribbon representations with the functional sites (223H-316D-355K in 1ord-A, 212E-214D-217E-228H in 1cel-A) represented as spheres. Each residue is colored by its predicted functional importance score, with the color changing from red to white to blue as the score decreases. For 1ord-A (A), both MFS and SeqonlyMFS work well in assigning the highest scores to the functional sites. However, ET and ConSurf also assign high scores to nearby residues in the surrounding cavity, thus the functional sites do not appear in the top-10 hits lists that are generated by these methods. For 1cel-A (B), all the sequence-based methods are able to assign relatively high scores to the functional sites (different shades of red color), but only the MFS method that uses structural information can boost the scores of the functional sites higher (more intense red color) to show up in the top-10 hits list.</p
Performance comparison of the MFS method, the SeqonlyMFS method (HMM_rel_ent+SSR+AAType), the Evolutionary Trace method, and the ConSurf method with the Thornton and Lovell datasets.
<p>Only proteins for which both the Evolutionary Trace and ConSurf methods are able to give predictions are used in the comparison. Four measures are used to compare the performance, including: ROC scores, the precision when sensitivity threshold is set at 20%, the false positive rate when sensitivity threshold is set at 20% and the top-10 hits. ET is only used in the ROC score computation but not in other comparative analysis, since it gives many tied scores for top-scoring residues. Both the MFS and SeqonlyMFS methods have better performances than methods that use only one type of information.</p
The application of MFS to understand the role of <i>btuba/btubb</i> dimer in the bacterial genus <i>Prosthecobacter</i> using the predicted and experimental structures.
<p>Both structures are colored by depicting higher MFS scoring residues with a more intense red color, with the top-10 high-scoring residues represented by spheres. One GTP and one GDP in the predicted structure, as well as one GDP and two SO<sub>4</sub><sup>2−</sup> ions in the experimental structure are shown as yellow spheres. The predicted structure is generated by homology-modeling techniques using the eukaryotic α/β tubulin dimer (PDB identifier: 1jff) as the template. The taxol ligand and metal ions are omitted from the predicted structure for easier depiction. In the predicted structure, <i>btubb</i> lies above <i>btuba</i>, with a GDP molecule enclosed by the dimer interface. In the experimental structure (PDB identifier: 2btq), <i>btuba</i> lies above <i>btubb</i> and there is no GDP in the dimer interface. Our MFS analysis first confirmed that <i>btuba</i> and <i>btubb</i> indeed form dimers due to the existence of a high-scoring cluster in their dimer interface, in contrast to previous predictions made by using the structural stability score alone. In addition, the MFS suggests that regardless of how <i>btuba</i> and <i>btubb</i> orient with each other, their interface is functionally important and may bind to GDP molecules.</p
Correlation coefficients of several components of the MFS method in the Thornton dataset (cells in upper-right triangle of the table) and the Lovell dataset (lower-left triangle), respectively.
<p>The components of the MFS method have a relatively low correlation with each other, demonstrating that they can provide complementary information toward accurate functional site prediction.</p
Prediction of residues with rare function not represented in the training sets.
<p>MFS was trained on a set of residues experimentally characterized to participate in canonical catalytic functionalities and protein-ligand interfaces. Protein binding to biomineral surfaces is a rare function and poorly understood process, for which the only diffraction structure available is osteocalcin binding metal ions (depicted as green spheres with ionic bonds to the γ-carboxy glutamic acid (gla) residues in transparent green tube) (PDB identifier: 1q8h). The three gla residues of osteocalcin (represented as spheres, similar to the target residues in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000181#pcbi-1000181-g003" target="_blank">Figure 3</a> above) previously shown to bind the hydroxyapatite surface of bone are clearly selected by MFS within the top six of 49 residues, with or without knowledge of structural and post-translational modification to these residues. These residues are selected within the top eight by ConSurf, with much lower discrimination from scores for the other residues in osteocalcin. None of these residues are selected within the top-10 by ET. This example demonstrates the applicability of MFS to make highly accurate and specific predictions for proteins of vastly diverse functions.</p
Sequencing results.
<p>Sanger and NGS sequencing coverage of targeted CRISPR mutations at the <i>pfatp4</i> locus for ACP-B6-L350H and ACP-B6-P412T with clonal wild type parent strain ACP-B6. Red bars delineate the respective 20 nt guide RNA target sites and PAM sites required for each edit. NGS coverage at each location is indicated by blue columns. (<b>a</b>) Sequencing data of targeted locus 1002–1072 in <i>pfatp4</i> from strain ACP-B6-L350H showing SJ733 resistance-conferring SNPs in L350 and four other synonymous mutations introduced by CRISPR. Sequences of wild type <i>pfatp4</i> and repair template ssODN L350H are shown in alignment. The two silent mutations in ssODN L350H located 39 and 42 nt away were not incorporated into ACP-B6-L350H. (<b>b</b>) Sequencing data of targeted locus 1206–1276 in <i>pfatp4</i> from strain ACP-B6-P412T showing the SJ733 resistance-conferring SNP and silent mutations introduced by CRISPR. Sequences of wild type <i>pfatp4</i> and repair template ssODN P412T are shown in alignment.</p
Strategy for introducing plasmid-free CRISPR/Cas9 edits to the <i>Plasmodium falciparum</i> gene <i>pfatp4</i>.
<p>Synchronized ring-stage parasites at 17% parasitemia in fresh donor RBCs were nucleofected with Cas9 protein, guide RNA, and template ssODN. Cultures were kept under drug pressure with 500 nM SJ733 starting on day two post transfection. After drug-resistant parasites emerged from culture, genomic DNA was isolated with standard phenol-chloroform extraction methods for library preparation. The presence and penetrance of the targeted CRISPR edits were confirmed using Sanger sequencing and whole genome NGS.</p
Plasmid-free CRISPR/Cas9 genome editing in <i>Plasmodium falciparum</i> confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733
<div><p>Genetic manipulation of the deadly malaria parasite <i>Plasmodium falciparum</i> remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite’s genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in <i>P</i>. <i>falciparum</i> without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in <i>P</i>. <i>falciparum</i> could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.</p></div
Characterization of drug resistance.
<p>Dose-response curves and EC<sub>50</sub> values for the antimalarial SJ733 on the parent strain ACP-B6 and the mutants ACP-B6-L350H and ACP-B6-P412T. The growth inhibition assay was conducted by seeding synchronized ring-stage parasites from each strain at 0.8% parasitemia in media supplemented with SJ733 at concentrations ranging from 3.16 nM to 100 μM and allowing for growth over 72 hours. Parasites were fixed with 1% paraformaldehyde and stained with 50 nM YOYO-1. Final parasitemia was assessed by flow cytometry and values were normalized to DMSO-only controls. Values reported are mean ± standard error (n = 3). The inset shows parasitemia of each culture after 72 hours of growth in the presence of DMSO only.</p
Self-Assembly of Filamentous Amelogenin Requires Calcium and Phosphate: From Dimers via Nanoribbons to Fibrils
Enamel matrix self-assembly has long been suggested as
the driving
force behind aligned nanofibrous hydroxyapatite formation. We tested
if amelogenin, the main enamel matrix protein, can self-assemble into
ribbon-like structures in physiologic solutions. Ribbons 17 nm wide
were observed to grow several micrometers in length, requiring calcium,
phosphate, and pH 4.0–6.0. The pH range suggests that the formation
of ion bridges through protonated histidine residues is essential
to self-assembly, supported by a statistical analysis of 212 phosphate-binding
proteins predicting 12 phosphate-binding histidines. Thermophoretic
analysis verified the importance of calcium and phosphate in self-assembly.
X-ray scattering characterized amelogenin dimers with dimensions fitting
the cross-section of the amelogenin ribbon, leading to the hypothesis
that antiparallel dimers are the building blocks of the ribbons. Over
5–7 days, ribbons self-organized into bundles composed of aligned
ribbons mimicking the structure of enamel crystallites in enamel rods.
These observations confirm reports of filamentous organic components
in developing enamel and provide a new model for matrix-templated
enamel mineralization