Computational protocol: Room temperature structures beyond 1.5 Å by serial femtosecond crystallography

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Protocol publication

[…] Methods are published earlier (). In brief: PYP micro-crystals were grown from highly concentrated PYP. The crystals were about 5 μm long needles with 2 μm diameter (Fig. ). The suspension (5 × 1011 crystals/ml) was jetted into vacuum by a gas dynamic virtual nozzle (). FEL X-ray pulses of 40 fs duration containing about 1011 photons/pulse were directed on the jet about 75 μm from the nozzle exit. The temperature of the jet was not controlled. However, since the jet velocity is about 10 m/s, the 75 μm path through the vacuum of the chamber until it is intercepted by the X-rays translates to about 7.5 μs. Assuming a cooling rate of 106 K/s, the jet cools about 7.5 K. Hence, the temperature at the X-ray FEL beam intersection can be estimated to be about 15 °C, which is close to ambient (room) temperature. Diffraction patterns were collected by a Cornell SLAC Pixel Area Detector (CSPAD) located 75.1 mm from the jet. The center of the CSPAC was electronically attenuated by about a factor of ∼7 to allow for an increase in the dynamic range that allows the simultaneous collection of low and high resolution reflections (Fig. ). With this setup, about 2.5 × 106 diffraction patterns (snapshots) were collected in the dark (Table ). Snapshots containing Bragg reflections were identified by CHEETAH (). Indexing and integration was done by CrystFEL (). The indexing ambiguity of the hexagonal PYP crystals was solved by methods () implemented in CrystFEL.Refinement was carried out with “REFMAC5” () using the “log-likelihood residual.” B-factors were averaged for individual amino acid residues by “MOLEMAN” () over the backbone atoms N, Cα, C, and O. These B-factors ⟨B⟩ind were plotted as a function of residue number. The trans/cis isomerization of the chromophore is controlled by three hydrogen bonds (Fig. ). Two extend from the phenolate oxygen (O4) of the chromophore head to Oδ of Glu-46 and Oη of Tyr-42, and the third hydrogen bond is formed by the carbonyl-oxygen of the chromophore foot and the peptide bond nitrogen of Cys-69. The lengths of the O4-Oδ and O4-Oη hydrogen bonds might depend also on the bond length of the chromophore ring C4' to the hydroxyl oxygen O4 (Fig. ), which is set to the relatively large value 1.362 Å by a restraint in the “REFMAC5” library file “mon_lib.cif.” If the X-ray data are weak and low resolution, this restraint enforces short hydrogen bonds. However, we tested this by setting the restraint to 1.238 Å, which is typically found in older “CNS” () and “XPLOR” () parameter files. This should bias the refinement towards longer hydrogen bonds. Refinement was carried out with either restraints (see below). The lengths of the hydrogen bonds were measured by the program “COOT” () and compared to those found within existing structures in the literature. […]

Pipeline specifications

Software tools REFMAC5, CNS, Coot
Application Protein structure analysis
Chemicals Hydrogen