In situ data collection and structure solution using the XtalCheck-S system

Lysozyme crystals were grown in an In Situ-1™ crystallization plate (MiTeGen) and screened using the XtalCheck-S, mounted on a XtaLAB Synergy-DW with a 4-circle kappa Universal Goniometer 2 and a HyPix6000-HE Hybrid Photon Counting detector. CrysAlisPro was used to imaging, and crystal object selection and data collection. The figure below shows the relevant CrysAlisPro GUI and images for the In Situ-1 crystallization plate.


Six crystal objects were selected for data collection and placed in the queue. Data were collected automatically for the series of objects without user intervention using Copper radiation. For each crystal, a total of 40° of data was collected using an exposure time of 20 seconds per 0.5° and 2θ = 22°. Of these, all exhibited protein-like diffraction up to ~1.8 Å. Following data collection, each scan was processed individually and then scaled together in CrysAlisPro. Afterwards, the data were passed through AIMLESS in CCP4i and the resulting statistics are reported in the table below. The data set is essentially complete to 1.8 Å, with good values for mean signal-to-noise ratio, Rmerge, Rmeas, and CC1/2. The completeness of the combined scans was 98.1% and 86.7% for all data and the highest resolution shell, respectively.

Unit cell lengths (Å)   79.55, 79.5, 37.9
Resolution (Å) (last shell)   28.09-1.80 (1.84-1.80)
# reflections / # unique   69674 / 11485
Completeness (%) (last shell)   98.1 (86.7)
Multiplicity (last shell)   6.1 (2.3)
<I>/<σ/(I)> (last shell)   17.3 (2.3)
Rmerg (%) (last shell)   5.8 (40.5)
Rmeas (%) (last shell)   6.3 / 49.6
CC1/2 (%) (last shell)   99.9 (77.8)

The structure was solved by molecular replacement using only the backbone of PDB ID 1LYZ as a search model in the phasing pipeline of HKL-3000 (which used MOLREP). The resulting MR solution was rebuilt using ARP/wARP and refined with REFMAC to produce a model with an R=15.0% and Rfree=19.5%. The anomalous difference map had strong peaks at the sulfur atom positions.

Electron density maps and model near Trp111. 2Fo-Fc after
refinement (blue mesh @ 2 rmsd), Fo-Fc (red and green meshes @ 3 rmsd),
and anomalous difference (magenta mesh @ 4 rmsd).