Computational protocol: Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

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Protocol publication

[…] Lethal eIF3 knock-out strains were validated by crossing heterokaryons obtained from the FGSC (see ) with wild-type Neurospora. Reciprocal crosses were done using the wild-type strain as either the male or female. Ascospores from each cross were harvested and germinated on Vogel’s minimal media, 2% agar, 2% sucrose supplemented with or without 200 µg/mL hygromycin B. No ascospores germinated on media containing hygromycin. Conidia harvested from germinated ascospores grown on non-hygromycin media subsequently failed to germinate when transferred to slants that contained Vogel’s minimal media, 2% agar, 2% sucrose supplemented with 200 µg/mL hygromycin B.Growth and conidiation phenotypes were assessed by spotting a small amount of frozen conidia in the middle of a plate containing Vogel’s minimal media, 2% agar and 2% sucrose. Plates were incubated at 30°C in the dark and photographed at the indicated times in .Race tubes were made using 25 mL serological pipettes. Autoclaved media (Sucrose media: Vogel’s minimal media, 2% agar, 2% sucrose or water agar: Vogel’s minimal media, 2% agar) was drawn up to the top of the pipette and expelled to the 13 mL line, at which point the pipette was laid down on a level surface until the media solidified. The tips of the pipettes were broken off and capped with a sterile cap right before spotting. Race tubes were spotted with conidia harvested using water from slants that had been grown at 30°C in the dark for 2–3 days and allowed to conidiate in the light for an additional 7–8 days. All strains within an independent race tube experiment used conidia of equal age. All race tube data were collected and analyzed in parallel with wild-type samples each time. After spotting, the race tubes were incubated at 30°C in the dark for 16–18 hours before taking the initial measurement. Subsequent measurements were typically taken in 6–8 hour increments over the course of 2–3 days. Linear growth was normalized to the first time point. Three technical replicates of each biological replicate were combined and plotted against time (hours) and fit with a linear function using Sigmaplot. The linear growth rate (slope) and errors on the slope of the linear fits were normalized as a fraction of the linear growth of wild-type. The presented data represent the combination of at least two independently isolated isogenic biological replicates, each with a minimum of three technical replicates. Knock-out strains transformed with empty vectors, which express only the HAT-FLAG affinity tag, had the same linear growth as their respective untransformed knock-out strain.For double knock-out and epistatic analysis, the predicted double knock-out linear growth phenotypes were calculated by multiplying the relative linear growth rates of the individual knock-outs. Predicted double knock-out errors were calculated using the root of the sum of squares of the standard errors of the individual knock-outs. [...] Protein samples were diluted to a final concentration of 50 nM in buffer 20 mM Hepes, pH = 7.4, 120 mM KCl, 0.5 mM EDTA, 1 mM DTT, and 3% trehalose. 400 mesh continuous carbon grids were plasma cleaned in a 75% Ar/25% O2 atmosphere for 20 seconds in a Solarus plasma cleaner (Gatan, Inc). Sample aliquots of ∼4 µL were placed onto the grids, negatively stained with a 2% uranyl acetate solution and blotted to dryness. Data were acquired using a Tecnai T12 electron microscope operating at 120 keV using the Leginon automated data collection software on a Tietz 4×4K pixel CCD camera (15 µm pixel size) at a nominal magnification of 50,000X (2.18 Å/pixel). Images were collected in low-dose mode with a dose of ∼20 e-/Å2 and a defocus range from −0.5 to −1.5 µm.Two-dimensional data processing was performed using programs and utilities contained within the Appion processing environment . Particles were initially extracted using a difference of Gaussians (DoG) particle picker . After contrast transfer function (CTF) estimation using CTFFind , particle image stacks were generated by extracting selected particles with a box size of 192×192 using the “batchboxer” program with an estimated CTF confidence cutoff above 80%. The data were then binned by a factor of two for processing. Each particle was normalized using the XMIPP normalization program using a cutoff of 4.5σ of the mean pixel value. The stack was subjected to several rounds of reference-free two-dimensional classification using IMAGIC iterative multivariate statistical analysis and multi-reference alignment analysis (MSA-MRA). Overlapping particles or dust were removed and only those classes belonging to properly assembled complexes were kept. A new stack was generated with all the particles within these classes and subjected to five final rounds of MSA-MRA. […]

Pipeline specifications

Software tools SigmaPlot, Leginon, Appion, CTFFIND, Xmipp, IMAGIC
Applications Miscellaneous, cryo-EM
Organisms Neurospora crassa, Homo sapiens