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Protocol publication

[…] The micrographs in set04 within the EMPIAR-10014 deposition (http://pdbe.org/empiar) from were used for analysis. Of the 581 images, the first 160 were used for filament extraction. The Contrast Transfer Function was measured using CTFFIND3 (). For the 160 images, the mean defocus was 3.2 μ and the range was from 1.7 μ to 5.4 μ. The images were then multiplied by the theoretical CTF function to correct the phases and improve the SNR. From these 160 images, 1066 long filament boxes were cut using the e2helixboxer routine within EMAN2 (). The SPIDER software package () was used for all further steps. Overlapping boxes 192 px long (2.3 Å/px) were cut from the long filament boxes using a shift of 6 px (97% overlap), generating 64,980 boxes. These were used for IHRSR () which converged to a symmetry of −101.2° rotation with an axial rise of 5.3 Å, starting from a solid cylinder as an initial reference. Due to the very poor resolution (∼17 Å), an attempt was made to improve this by only using the 58 images with a defocus less than 3.0 μ, which yielded 15,624 overlapping boxes. Because segments with a very large out-of-plane tilt were excluded, the final reconstruction was generated from 8600 segments. The modulation of the amplitudes of the final reconstruction by the CTF (the phase reversals of the CTF were previously corrected when the images were multiplied by the CTF) was corrected by dividing the Fourier coefficients in the reconstruction by the sum of the squared CTFs, imposing a negative B-factor of 2000 to correct for the decay of the high frequencies, and filtering to 12 Å. […]

Pipeline specifications

Software tools CTFFIND, EMAN, SPIDER
Databases PDBe EMPIAR
Diseases Wiskott-Aldrich Syndrome