Computational protocol: Femtosecond X-ray diffraction from two-dimensional protein crystals

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Protocol publication

[…] Owing to the use of a non-tilting sample stage, diffraction patterns were assumed to be untilted and normal to the X-ray pulse. Matlab, CCP4 and UCSF Chimera were used for all analysis. Initially, the diffraction patterns were overlaid with expected lattice positions calculated from assumed unit-cell parameters of a = b = 82 Å and α = β = γ = 90° with C 222 symmetry for streptavidin and a = b = 63 Å and α = β = 90°, γ = 120° with P 3 symmetry for bacteriorhodopsin. These unit-cell parameters were derived from both transmission electron microscope data of similarly prepared samples and from previous publications (Darst et al., 1991, Henderson et al., 1990, Lenne et al., 2000, Verclas et al., 1999). For both samples the expected lattice unit-cell parameters for an untilted crystal closely matched the observed reflections and were used as a local marker for subsequent peak searches. It should be noted that in the case of streptavidin, the C 222 symmetry results in systematic absences for reflections where the indices h + k = 2n + 1, so the innermost spots within the streptavidin lattice represent the (0, 2), (1, 1) and (2, 0) reflections not (0, 1), (1, 1) and (1, 0). Localized peak searches were then performed using the expected lattice positions as the central starting locations and only peak values equal to or greater than 7 ADU (analog-to-digital units) were considered valid peaks since a single 8.4 keV photon yields a pixel value of approximately 8 ADU on the CSPAD. The integrated intensity for each identified peak was calculated by using a 3 × 3 pixel search to identify all connected pixels with values greater than 7 and summing them together with the central peak. Only integrated intensities greater than 15 were included in subsequent analysis since this provided a signal-to-background ratio of at least 5. To estimate the electron density projection map from the observed reflections of each single-shot pattern, a generalized molecular replacement scheme was used, wherein the CCP4 program SFALL was first used to generate a list for all calculated reflections (H, K, L, F c and phase) to 8 Å resolution from the known structures of bacteriorhodopsin (PDB: 2ntu) and streptavidin (PDB: 3rdx). A final experimental reflection list was created by combining the H, K, L and corresponding integrated intensity for each experimentally observed spot with the calculated phase (PHIC) values generated by SFALL for the corresponding calculated reflections. The CCP4 program F2MTZ then converted the experimental reflection lists from HKL to MTZ format and the resulting spot lists were input into the CCP4 program FFT to yield a 2-D electron density projection map of a 2 × 2 unit cell. UCSF Chimera was used to visualize the 2 × 2 unit-cell ribbon diagrams of the known structures for comparison. […]

Pipeline specifications

Software tools CCP4, UCSF Chimera
Applications cryo-EM, Protein structure analysis
Organisms Dipturus trachyderma