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

    Application Note PX022


    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.

    X-ray source PhotonJet-S Cu source with continuously variable divergence slit
    Beam size = 100 µm
    Operating power 50 kV x 1 mA = 50 W
    Goniometer / Detector range 4- circle Kappa with telescoping 2θ arm / distance range of 30 – 250 mm
    Active area
    Readout time
    Pixel size
    Hybrid photon counting HyPix-6000HE
    77.5 x 80.3 mm 
    Continuous (7 ns)
    100 µm

    Data collection was performed using CrysAlisPro, 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).

    PX022 Figure 1 Thaumatin crystal

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


    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).

    PX022 Figure 2 Diffraction image

    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 CrysAlisPro, were collected to achieve complete, redundant data to 1.4 Å in 2 hours (Table 2).

    Table 2: Scans collected on thaumatin.

    Scan # ω start ω range Δω φ κ No. img Time/img Total exposure
    1 -136° 186° 0.15° -180° -37.8° 1240 2s 41m 20s
    2 55° 37.95° 0.15° 84° -171° 36.9° 253 2s 8m 26s
    3 60° 64.05° 0.15° -150° 178° 36.9° 427 2s 14m 14s
    4 -27° 27° 0.15° -97.7° 112° 36.9° 180 2s 6m
    5 -119° 118.05° 0.15° 30° -57° -37.8° 787 2s 26m 14s
    6 -58° 108° 0.15° -30° -19° -37.8° 720 2s 24m
      2h 14s

    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 Rmerge under 10%. To locate the 17 sulfur sites and phase the data, SHELXD³ was run for 500 trials and the best solution had a CCAll = 25.1, CCWeak = 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 Rfree =  16.2%. Figure 3 shows the model and maps near R53.

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

    Space group P4₁2₁2
    Unit cell (Å) 57.8, 57.8, 149.8
    Resolution (Å) (last shell) 20-1.4 (1.44-1.40)
    Total # reflections 698447
    Unique # reflections 95481
    Completeness (%) (last shell) 99.9 (100)
    Multiplicity (last shell) 7.3 (5.2)
    <I/σ(I)> (last shell) 19.6 (1.7)
    Rmerge (%) (last shell) 7.3 (88.8)
    CC½ (last shell) 99.9 (57.7)
    R / Rfree (%) 15.0 / 16.2

    PX022 Figure 3 electron density maps

    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.


    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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