Serial data collection for trypsin crystals

Bovine pancreas trypsin was screened for crystallization conditions using the Rigaku Reagents Wizard Classic 1 & 2 screens in a MiTeGen In Situ-1™ crystallization plate. Following an incubation period at 18°C, the plate was loaded into the XtalCheck hardware, which was mounted on a XtaLAB MM007-HF diffraction system configured with the MicroMax™-007 HF microfocus rotating anode, a VariMax HF optic and a PILATUS3 R 200K Hybrid Photon Counting detector.

Data were collected for six different trypsin crystals located in a crystallization drop in well H5. For each crystal, a total of 43 degrees of data were collected using an exposure time of 3 seconds per 0.25°. Following data collection, the data were processed using HKL-3000R1. Each crystal indexed with similar unit cell parameters in a primitive orthorhombic lattice.

Each data scan was integrated separately, then all scans were scaled together. Resulting data processing statistics are shown in Table 1. The data merge together well with completeness of 96% and 84.9% for all data and the highest resolution shell, respectively. The R-symm and R-meas are 2.9% and 5.4%, respectively. Scaling revealed the resolution limit for all data to be beyond 2.0 Å given that the / was much greater than 2 at the edge of the detector.

Space Group   P2₁2₁2₁
Avg. unit cell lengths   54.91 Å, 58.62 Å, 67.49 Å
Resolution   1.96 Å
Mosaicity range   0.29° - 0.42°
# reflections / # unique   79840 / 16227
# reflections rejected / %   364 / 0.46
Redundancy (last shell)   5.1 (27)
Completeness (last shell)   96.% (84.9%)
<I>/<σ/(I)> (last shell)   84.9 (6.7)
Merged R-symm / R-meas   2.9% / 5.4%
Chi² (last shell)   1.119 (1.972)

HKL-3000R was used to run third party programs for structure solution by molecular replacement, including CCP4, ARP/wARP and Coot. Prior to molecular replacement, the 1BTY PDB file was edited to remove side chains, water molecules and ligands. Then, MOLREP was run using a resolution limit of 3.0 Å to place a single chain of the search model. The structure was automatically rebuilt using 3 cycles of ARP/wARP. The resulting model contained residues 7 through 224 with an R = 17.0% and Rfree = 22.5%. Then, 10 cycles of refinement were performed with Refmac to reach a final R = 15.8% and Rfree = 21.4%. Figure 4 shows the 2Fo – Fc map with the model.

2Fo-Fc electron density map (blue) contoured at 1σ and refined to 2.0 Å,
shown with overlay structure autobuilt by ARP/wARP and refined by Refmac.