X-ray data collection, structure determination, and refinement
This protocol is extracted from research article:
Structure and genome ejection mechanism of Staphylococcus aureus phage P68
Sci Adv, Oct 16, 2019; DOI: 10.1126/sciadv.aaw7414

Crystals of tail fiber protein in mother liquor solution were vitrified by plunging into liquid nitrogen. Diffraction data were collected at the SOLEIL synchrotron on beamline PROXIMA-1 equipped with a Pilatus 6M detector. The data were integrated and scaled using the package XDS (55). The model for molecular replacement was prepared by converting the tail fiber of phage phi11 (PDB 5EFV) to poly-alanine and rebuilding it according to the threefold averaged cryo-EM map of the P68 tail fiber (56). The quality of the cryo-EM map was sufficient for building the poly-alanine chain of residues 150 to 570, except for several loops in poorly resolved regions of the map. Molecular replacement was performed using the program Phaser (57). The trimers of the tail fiber proteins sit on threefold axes of the R3 space group. Because of the crystal packing, the stem domain has to be bent nonphysiologically within the arm region. It is likely that the bending of the arm region differs among subunits within the crystal, which results in a local disorder. Therefore, the x-ray electron density map does not contain resolved features corresponding to the arm domain of the tail fiber protein. The structure was subjected to several rounds of automated building using the programs Phenix_auto_build and ARP/wARP combined with manual corrections in COOT and refinement in REFMAC5 (52, 54, 58).

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