r ggmsa package  (RStudio)

Journal: eLife

Article Title: On the role of VP3-PI3P interaction in birnavirus endosomal membrane targeting

doi: 10.7554/eLife.97261

Figure Lengend Snippet: ( A , left panel). Immunoblots of the top (T) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-2xFYVE protein (~35 kDa) specifically binds to liposome PI3P(+). The bar plot represents the intensity of T/B bands for each liposome preparation. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( A , right panel). Immunoblots of the three top (T 1 , T 2 , and T 3 ) and bottom ( B ) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 FL R 200 D protein (~35 kDa) does not bind to liposome PI3P(-) nor PI3P(+). The bar plot represents the intensity of (T 1 +T 2 +T 3 )/B bands for each liposome preparation. Significant differences (ns p>0.05) as determined by one-way ANOVA with Tukey’s HSD test. ( B ) Far-UV CD spectra of His-VP3 FL (red line) or His-VP3 FL R 200 D (green line). Spectral acquisitions at 50 nm/min with 0.1 nm steps at 1 s integration time, with a bandwidth of 1 nm were performed four times for the samples as well as for the buffer. The measurements were carried out with constant nitrogen gas flux of 10 ml/min. Acquisitions were averaged and buffer baseline was subtracted with Spectra Manager (JASCO). No smoothing was applied. CDtoolX was used to zero between 255–260 nm and to calibrate the signal amplitude from the fresh CSA signal . Data are presented as delta epsilon (Δε) per residue (L.mol- 1 .cm- 1 .residue 1 ) calculated using the molar concentration of protein and number of residues. ( C ) QM7 cells were grown in M24 multi-well plate for 12 hr to approximately 90–95% confluency and then 800 ng of plasmids were transfected [(SegA +SegB) or (SegA.R 200 D+SegB)] in triplicate. At 8 hr post-transfection (p.t.) the supernatants were discarded, and the monolayers were recovered for further plating on M6 multi-well plates containing non transfected QM7 cells. Avicel RC-591 (FMC Biopolymer) was added to the M6 multi-well plates. 72 hr p.i., the monolayers were fixated and stained with Coomassie R250 for revealing the foci forming units. ( D ) Partial view of amino acid alignment of the VP3 protein for nine reference members of Birnaviridae family. Multiple sequence alignment was performed with Clustal OMEGA (v1.2.4) implemented at EMBL’s European Bioinformatics Institute (The complete alignment is shown in ). Alignment visualization was done with the R ggmsa package in with assistance from the RStudio software . Amino acids are colored according to their side-chain chemistry. Protein sequence logos annotation is displayed on top of the amino acid alignment. For facilitating the view IBDV VP3 142–210 portion is shown. The black arrow indicates the K 157 , R 159 , H 198 , and R 200 . Figure 4—source data 1. Original membranes corresponding to . Figure 4—source data 2. Individual files corresponding to the original membranes from .

Article Snippet: Alignment visualization was done with the R ggmsa package ( ) with the assistance from the RStudio software ( ).

Techniques: Western Blot, Circular Dichroism, Residue, Concentration Assay, Transfection, Staining, Sequencing, Software