Computational protocol: Computational and genetic evidence that different structural conformations of a non-catalytic region affect the function of plant cellulose synthase

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Protocol publication

[…] Secondary structure predictions were generated using Psipred () and the topology prediction of the TMH region was obtained with the OCTOPUS software utility (). Lipophilicity scoring was used to further constrain the predictions (), and additional constraints for Rosetta were obtained from the JUFO utility (). Starting coordinates of the amino acid sequence in linear form were generated using the tleap utility of the molecular dynamics software suite AMBER. An in-house script cleaned the resulting coordinate file to be compatible with ROSETTA atom naming conventions.Three-dimensional structural decoys of the TMH5–6 regions of native and mutant CESAs were generated using the membrane protein folding algorithm of ROSETTA (ver 3.4) () (see Supplementary Table S1 for details). The ROSETTA fragment library database used was generated using the 2010 NCBI non-redundant protein database. Optimal structures were relaxed using the ROSETTA ab initio relax method under a weak constraint. As the decoys resulting from production runs did not form a clear folding plot that converged on a low-REU decoy, clustering analysis was employed to select decoys predicted to be near-native (energetically stable) structures. The Durandal algorithm and maximum-entropy based clustering of the top ten percent best scoring decoys were used for the selection of model structures ().Origin (OriginLab, Northampton, MA) or Tecplot (Tecplot Inc., Bellevue, WA) were used to generate the folding plots and PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC) and Discovery Studio (Accelrys Software Inc., San Diego, CA) were used to view and render protein structures. Additional folding plots were generated using a custom shell script and the Durandal software utility that calculated the C-alpha root-mean-square deviation (CARMSD) between all generated decoys against the best scoring structure. ROSETTA does have a scoring utility, but it is significantly slower than Durandal. To obtain decoy densities, frequency counts needed for heatmaps were generated by binning in areas of 0.5 REU by 1 Å CARMSD. Structures of the region of BcsA corresponding to the TMH5–6 region of plant CESA were taken from the crystal structure (PDB ID: 4HG6) and rendered with PyMOL. A custom batch script that leveraged local installations of all the required utilities and topology prediction engines except for JUFO was created to accomplish this process of linear sequence to initial analysis and automated structure selection. [...] Seeds were grown on vertical plates at 23 °C (permissive temperature) for 5 d, then transferred to 31 °C (restrictive temperature) for an additional 7 d. Seedlings were grown under continuous light of approximately 100 µmol m–2 s–1 and 50% relative humidity. Primary roots were photographed then analysed using ImageJ (http://rsbweb.nih.gov/ij/). The means for root length were derived from measurements of 10–30 individual seedlings per line from at least two independent experiments. [...] Atcesa6 prc1 Atcesa1 rsw1 YFP–CESA6 and Atcesa6 prc1 Atcesa1 rsw1 YFP–CESA6 Atcesa1 F954L seeds were surface-sterilized in 30% bleach + 0.1% (w/v) SDS for 20min, washed four times with sterile water, stored at 4 °C for 2 d, and sown on half-strength Murashige and Skoog medium without sucrose. Plates were exposed to light for 2h to stimulate germination, then wrapped in two layers of aluminium foil and grown vertically for 3 d at 22 °C. For restrictive temperature treatment, plates with three-day-old etiolated seedlings were transferred to a 29 °C incubator for 24h. For all genotypes, YFP fluorescence was detected in hypocotyl cells just below the apical hook using a 100 mW 488nm excitation laser at 30% power and a 525/50nm emission filter on a Zeiss Observer SD spinning disk confocal microscope with a ×100 1.40 NA oil-immersion objective and a Photometrics QuantEM 512SC camera (exposure time=400ms, EM gain=1000, readout gain=1). YFP–CESA6 particle density for z series was quantified computationally using the Spot Detection function of Imaris (Bitplane) (). For fluorescence intensity measurements, z series were opened in ImageJ, extraneous z slices were removed using the Substack Maker plugin, maximum projections were generated and background subtracted using a sliding paraboloid radius of 10 pixels, and Integrated Density for the entire image was measured. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Arabidopsis thaliana