S-SAD phasing of lysozyme using PILATUS3 R 1M

This example illustrates data collection and S-SAD phasing of lysozyme using the PILATUS3 R 1M, a hybrid pixel array detector. The PILATUS3 R 1M can be offered as a drop-in replacement for existing R-AXIS detectors, for those labs that want a large format detector with faster throughput.

Data were collected for a lysozyme crystal on the XtaLAB PRO configured with an FR-X microfocus rotating anode generator for a total of 6 minutes.

Following scaling, the overall redundancy was 11.9. The structure was easily solved using S-SAD method in HKL-3000R. HKL-3000R used SHELXC and SHELXD to find sites using data to 2.0 Å resolution.

Fourteen sites were anomalous sites were located with occupancies greater than 0.6. Following phasing and density modification using MlPhare and DM the FOM was 0.77. Five rounds of ARP/wARP were used to trace the protein with 96% of the atoms correctly placed. Refmac was used to refine the structure with some manual model adjustments and addition of the remaining residues and water molecules. The final R and Rfree for the structure were 15.1% and 20.1%, respectively. Additional details of the processing and structure solution are provided below.

Degrees of data collected 180
Exposure time 1 sec
Space group P4₃2₁2
Resolution 1.76
Total # reflections (unique) 142494 (12016)
# reflections rejected / % 14 / 0.01
Completeness (%) (last shell) 99.7 (96.9)
Redundancy (last shell) 11.9 (8.7)
<I>/<σ(I)> (last shell) 65.0 (15.7)
Linear R-merge (%) 3.5
Χ² (last shell) 0.86 (0.66)
Resolution for heavy atom search 2.0
# sites found 14
Correlation coefficients (all/weak) 36.17/18.73
FOM after DM 0.77
# autobuild cycles/% residues autobuilt 5/96
# refinement cycles 10
R/Rfree after refinement (FOM) 15.1/20.1 (0.893)