Computational protocol: Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores

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Protocol publication

[…] Thin-section EM was performed as previously described (). Cells were grown to early log phase at 30°C in SRC-Leu and then shifted to 2% galactose for 10 h. Negative staining for single-particle EM was performed as described previously (). Micrographs were recorded with a field emission gun microscope (CM-200; Phillips) under low dose conditions on a 2K × 2K charge-coupled device camera (TVIPS F224; Tietz) at 200 kV with a nominal magnification of 38,000× (calibrated pixel size, 3.76 Å/pixel) and 50,000×, respectively (calibrated pixel size, 2.86 Å/pixel). For image processing, a total of 4,000 (for Nup170) and 1,500 (for Nup170C) particle images were selected and boxed using the Medical Research Council image-processing package () or the SPIDER software package (). All subsequent image processing was performed in Imagic V (Image Science Software GmbH; ). Alignment, iterative refinement of class averages, and the calculation of the 3D maps followed previously described procedures ().Fold assignment of Nup170 was performed as previously described (). Secondary structure prediction of Nup170 was performed using psipred (), and the 3D model was built by modeller-8 () from the hhsearch () alignment. […]

Pipeline specifications

Software tools SPIDER, IMAGIC, PSIPRED, MODELLER, HHSearch
Applications Protein structure analysis, Amino acid sequence alignment
Organisms Saccharomyces cerevisiae