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    ClpS SAD phasing with 30 Minutes of Data Collection on the XtaLAB Synergy-S

    Application Note PX023

    Introduction

    Rigaku’s XtaLAB Synergy-S is much more than a crystal screening instrument. It comes equipped with a powerful source, an ultrafast kappa goniometer, and a hybrid photon counting detector to enable fast data collections on macromolecular crystals. Here, we show a 30-minute data collection on an orthorhombic crystal of ClpS. The data are complete to 1.28 Å and the structure can be solved by single wavelength anomalous dispersion (SAD).

    Experimental overview

    Cryocooled crystals of ClpS were provided by the laboratory of Karl Schmitz (University of Delaware). The crystallization condition was 10% PEG3350, 7.5 mM NiCl₂, 0.1 M HEPES pH 7.5. The PEG3350 was increased to 40% before flash-cooling in liquid nitrogen. 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
    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
    Detector
    Active area
    Readout time
    Pixel size
    Cooling    		 Hybrid photon counting HyPix-6000HE
    77.5 x 80.3 mm 
    Continuous (7 ns)
    100 µm
    air-cooled

    The crystal was transferred from liquid nitrogen storage dewar to the goniometer using cryotongs. The crystal was kept at 100 K using an Oxford Cryosystems Cryostream 800. Data collection and processing were performed using CrysAlisPro, and file preparation and structure solution were performed using various programs of CCP4¹. A large 0.25 mm x 0.3 mm x 0.4 mm crystal was used for data collection (Figure 1).

    PX023 Figure 1 ClpW crystal

    Figure 1: ClpS crystal. Grid spacing is 0.1 mm. Circle is 0.1 mm.

    PX023 Figure 2 diffraction images

    Figure 2: 0.8-second exposure per 0.25° rotation of the ClpS crystal at distance of 32 mm and 2θ of 22.8° on the HyPix-6000HE. The resolution rings from left to right are 4.56, 2.28, 1.64, and 1.38 Å. Zoomed panel shows weak, high resolution spots.

    The ClpS crystal was screened using a crystal to detector distance of 32 mm and 0.8 second exposures per 0.25° rotation. The highest resolution diffraction spots were visible to ~1.45 Å (Figure 2). Autoindexing revealed a primitive, orthorhombic unit cell with a = 51 Å, b = 52 Å and c = 54 Å. Ten scans, calculated by the strategy algorithm of CrysAlisPro, were collected to achieve complete, 9-fold redundant data to 1.3 Å in 30 minutes (Table 2).

    Table 2: Scans collected on the ClpS crystal.

    Scan # ω start ω range Δω φ κ 2θ No. img Time / img Total exposure
    1 -58° 61° 0.25° -152° 105° -23.35° 244 0.8s 3m 15.2s
    2 -48° 91° 0.25° 118° 86° -23.35° 364 0.8s 4m 51.2s
    3 -32° 81° 0.25° -10° 41° -23.35° 324 0.8s 4m 19.2s
    4 2° 26° 0.25° -120° 77° 22.8° 104 0.8s 1m 23.2s
    5 2° 26° 0.25° -180° 77° 22.8° 104 0.8s 1m 23.2s
    6 2° 26° 0.25° 120° 77° 22.8° 104 0.8s 1m 23.2s
    7 -1° 55° 0.25° -60° -99° 22.8° 220 0.8s 2m 56s
    8 -51° 79° 0.25° -150° -19° 22.8° 316 0.8s 4m 12.8s
    9 -28° 72° 0.25° 0° -77° 22.8° 288 0.8s 3m 50.4s
    10 -93° 45° 0.25° 60° -178° -23.35° 180 0.8s 2m 24s
      29m 58s

    Table 3: ClpS processing statistics.

    Space group P2₁2₁2₁
    Unit cell (Å) 51.5, 52.2, 54.7
    Resolution (Å) (last shell) 24.2-1.28 (1.30-1.28)
    Total # reflections 360644
    Unique # reflections 38669
    Completeness (%) (last shell) 99.8 (97.2)
    Multiplicity (last shell) 9.3 (4.7)
    <I/σ(I)> (last shell) 22.0 (1.9)
    Rmerge (%) (last shell) 4.9 (75.1)
    Rmeas (%) (last shell) 5.2 (84.2)
    Rpim (%) (last shell) 1.6 (36.5)
    CC½ (last shell) 1.0 (0.63)
    R / Rfree (%) 12.9 / 16.3

    The resulting data were integrated and scaled to a limit of 1.28 Å where the last shell had 97% completeness, a suitable mean signal-to-noise ratio of 1.9, and a high C½ of 0.63 (Table 3). This resulted in a 99% complete data set with an Rmerge of 5%. The data had a small but measurable anomalous signal above the level of noise (0.8) to ~2 Å as reported by SHELXC² (Table 4). Using the Matthews coefficient, the solvent content was estimated at 39% with two molecules of ClpS in the asymmetric unit. Therefore, the potential anomalous scatterers were the 4 sulfurs per ClpS chain (only in Met residues) and any ordered Ni ions from the crystallization condition. To try to locate the 8 sulfur sites and phase the data, SHELXD² was run to 2.2 Å for 4000 trials and the best solution had a CCAll = 31.0, CCWeak = 16.8, CFOM = 47.8, and 11 sites with high occupancy. The 11 sites (8 sulfurs and 3 Ni ions shown in Figure 3) were used in phasing to 1.28 Å with the PHASER SAD pipeline (PHASER³, PARROT⁴, BUCCANEER⁵). The resulting model was subjected to several rounds of adjustment in COOT⁶ and refinement with REFMAC⁷ to give an R = 12.9% and an Rfree =  16.3%. A total of 156 residues were modeled, residues 5 to 80 in chain A and 1 to 80 in chain B (Figure 3).

    Table 4: Anomalous signal to noise ratio per resolution shell.

    Shell Mean anomalous signal to noise
    37.75 – 6.86 1.27
    6.86 – 4.22 1.25
    4.22 – 3.18 1.11
    3.18 – 2.60 0.93
    2.60 – 2.22 0.88
    2.22 – 1.96 0.89
    1.96 – 1.76 0.84
    1.76 – 1.60 0.78
    1.60 – 1.47 0.77
    1.47 – 1.37 0.75
    1.37 – 1.28 0.75

    PX023 Figure 3 Cartoon ribbon of ClpS

    Figure 3: Cartoon ribbon of the 2 molecules of ClpS in the asymmetric unit (chain A is green and chain B is blue). All 8 Met residues are shown in stick representation. The anomalous difference map (orange) is contoured at 5 rmsd, revealing 11 peaks.

    Results

    Interestingly, the three well-ordered Ni²⁺ ions identified by their anomalous peaks appear to promote intermolecular interactions between crystallographic symmetry mates of  ClpS. The Ni²⁺ ions were coordinated in octahedral geometry with water molecules and at least one side chain (a His or Glu) in two different molecules of ClpS (Figure 4). Given that ClpS functions as a monomer in solution, the contacts appear to be a result of the crystallization process.

    PX023 Figure 4 electron density

    Figure 4: 2Fo-Fc (blue mesh @ 2 rmsd), Fo-Fc (green and red meshes @ 3 and -3 rmsd, respectively), and anomalous difference (orange mesh @ 5 rmsd) electron density maps. Strongest Ni²⁺ ion peak is shown at center.

    Conclusion

    A high-resolution, 1.28 Å data set was collected on a ClpS crystal in just 0.5 hrs using the XtaLAB Synergy-S. The data were of superior quality as evidenced by the data processing statistics and even more so by the success of SAD phasing—despite a small anomalous signal and multiplicity of less than 10 overall. Clearly, a modern microfocus sealed tube diffractometer can be the workhorse instrument for the home laboratory.  

    References

    1.  Winn, M.D. (2011) Acta Cryst. D67, 235-242.
    2. Sheldrick, G.M. (2008) Acta Cryst. D64, 112-122. 
    3. Read, R.J. & McCoy, A.J. (2011) Acta Cryst. D67, 338-344. 
    4. Cowtan, K. (2010) Acta Cryst. D66, 470-478.
    5. Cowtan, K. (2006) Acta Cryst. D62, 1002-1011.
    6. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. (2010) Acta Cryst. D66, 486-501.
    7. Murshudov G.N., Vagin, A.A. & Dodson, E.J. (1997) Acta Cryst. D53, 240-255.

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