Thaumatin S-SAD Phasing with 2 hours of Data Collection on the XtaLAB Synergy-S

Introduction

We recently introduced a sealed-tube 4-circle diffractometer for small molecule and macromolecular crystallography, the XtaLAB Synergy-S. It features three new technologies in a compact cabinet that will fit into any home laboratory: microfocus PhotonJet-S sources (with Cu, Mo, or Ag targets), an ultrafast (10° per second) Kappa goniometer, and a HyPix-6000HE hybrid photon counting detector with 0.1 mm by 0.1 mm pixels. Here, we collected a two-hour data set on a thaumatin crystal to demonstrate the quality of data from the XtaLAB Synergy-S system using a protein crystal with an average size unit cell.

Experimental overview

T. daniellii thaumatin was purchased from Sigma and suspended in ultrapure, laboratory grade water to a concentration of 50 mg/ml. Crystals of thaumatin were grown at 18˚C from hanging drops consisting of 6 μL of 50 mg/ml thaumatin plus 4 μL of crystallization solution [24% (w/v) NaKTartrate, 15% (v/v)  ethylene glycol, and 0.1 M BisTris Propane, pH 6.6] over a well containing 500 μL of crystallization solution. Prior to flash-cooling, each crystal was soaked for ~15 to 30 seconds in the above crystallization solution at 25% (v/v) ethylene glycol for cryoprotection. The XtaLAB Synergy-S system and detector specifications are listed in Table 1.

Table 1: XtaLAB Synergy-S specifications.

Data collection was performed using CrysAlis^Pro, processing was performed using XDS¹, and structure solution was performed using various programs through HKL-3000R². A large (0.6 mm x 0.3 mm x 0.3 mm) thaumatin crystal was selected for data collection to illustrate the worst-case scenario for spot spread due to beam divergence (Figure 1).

Figure 1: Thaumatin crystal. Grid spacing is 0.1 mm. Circle is 0.3 mm.

Results

The thaumatin crystal diffracted to beyond 2 Å and auto-indexing revealed a primitive, tetragonal unit cell a = b = 57 Å and c = 149 Å. Spot separation along the longest primitive cell edge was achieved using the full divergence of the X-ray beam and a detector distance of 60 mm (Figure 2).

Figure 2: 20-second exposure per 0.2° rotation of the thaumatin crystal at distance of 60 mm on the HyPix-6000HE. The resolution rings are 7.09, 3.74, and 2.68 Å. Zoomed in panel shows lunes along the 150 Å cell edge. Peak separation and d-spacing of spots boxed in blue is shown in the plot at right.

Six scans, calculated by the strategy algorithm of CrysAlis^Pro, were collected to achieve complete, redundant data to 1.4 Å in 2 hours (Table 2).

Table 2: Scans collected on thaumatin.

Table 3 shows the results of data processing. The data were scaled to 1.4 Å treating the Friedel pairs as different reflections because the high redundancy allowed it. This resulted in a 100% complete data set with an R_merge under 10%. To locate the 17 sulfur sites and phase the data, SHELXD³ was run for 500 trials and the best solution had a CC_All = 25.1, CC_Weak = 11.4, PATFOM = 0.93, and 14 sites with high occupancy. The 14 sites were used in phasing to 1.5 Å with SHELXE, refinement of sites and phase improvement with MLPHARE⁴, and density modification and phase extension to 1.4 Å with DM⁵. The Figure of Merit after density modification was 0.87. From the anomalous difference Fourier, 2 additional sulfur sites were picked up and the phasing process repeated. The phases were handed to ARP/wARP⁶ for 2 cycles of automated model building which produced an initial model containing 205 aa. This model was manually adjusted in Coot⁷ and refined for 10 cycles with Refmac⁸ which gave an R = 15.0% and an R_free =  16.2%. Figure 3 shows the model and maps near R53.

Table 3: Crystal parameters and processing statistics for Thaumatin with Friedel pairs unmerged.

Figure 3: 2Fo-Fc (grey mesh @ 2 rmsd), Fo-Fc (green and red meshes @ 3 and -3 rmsd, respectively), and anomalous difference (magenta mesh @ 5 rmsd) electron density maps. R53 is in the center flanked by two disulfide bonds.

Conclusion

The crystal structure of T. daniellii thaumatin was solved by S-SAD phasing to 1.4 Å using a data set collected in 2 hours on the XtaLAB Synergy-S system. This solution was possible in such a short time because of the high flux and brilliance of the system combined with the speed, accuracy, and precision of the ultrafast goniometer and HyPix-6000HE detector.

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