Mosquito crystallization robot

YfcM was concentrated by ultrafiltration to 15.7 mg ml⁻¹. The 6.3 mg ml⁻¹ YfcM solution was used for crystallization screening. Crystallization of YfcM was performed by the sitting-drop vapour-diffusion method at 293 K. A Mosquito crystallization robot (TTP Labtech) was used to mix 100 nl protein solution with 100 nl reservoir solution in a 96-well VIOLAMO crystallization plate (AS ONE). The following crystallization screening kits were used: Crystal Screen, Crystal Screen 2, Index (Hampton Research), Wizard I, Wizard II (Emerald Bio), MemGold (Molecular Dimensions), The PACT Suite and The JCSG+ Suite (Qiagen). However, even small crystals were not generated under these conditions. We then used the in situ proteolysis crystallization method (Dong et al., 2007 ▶ ). The 7.9 mg ml⁻¹ YfcM solution was incubated with a small amount of trypsin [1:100(w:w)] and used for crystallization screening in the same manner as above. Under these conditions, small crystals were generated under several conditions containing PEG in 1 d. Initial crystallization conditions were optimized by varying the concentrations of salt and PEG. Furthermore, the size and shape of the crystals were improved by the hanging-drop vapour-diffusion method. Crystals were grown in 4 µl drops prepared by mixing 2 µl protein solution and 2 µl reservoir solution. Crystallization information is given in Table 2 ▶.

Aliquots of the purified proteins were set up for crystallization using a mosquito^® crystallization robot (TTP Labtech). Coarse screens were typically setup onto Greiner 3-well plates using three different drop ratios of precipitant to protein per condition (100+50 nL, 75+75 nL and 50+100 nL). All crystallizations were carried out using the sitting drop vapour diffusion method at 4°C and were crystallized as described [14 (link)]. Crystals were cryo-protected using the well solution supplemented with additional ethylene glycol and were flash frozen in liquid nitrogen. Data were collected at diamond beamline I04 at a wavelength of 1.0121 Å. Indexing and integration was carried out using MOSFLM [53 ] and scaling was performed with SCALA [54 ]. Initial phases were calculated by molecular replacement with PHASER [55 (link)] using available structures of wild type proteins [14 (link)]. Initial models were built by ARP/wARP [56 (link)] and building was completed manually with COOT [57 (link)]. Refinement was carried out in REFMAC5 [58 (link)]. Thermal motions were analyzed using TLSMD [59 (link)] and hydrogen atoms were included in late refinement cycles.

Crystals of the A. pompejana DBD were grown at 20 °C with the sitting drop vapor diffusion technique using a Mosquito® crystallization robot (TTP Labtech) and SWISSCI 3-lens crystallization plates. Protein solution: 7.2 mg/ml in 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM TCEP. Crystallization buffer: 22% PEG 3350, 0.2 M MgCl₂, 0.1 M Tris, pH 8.3. Drop size: 300 nL (150 nL protein solution + 150 nL crystallization buffer). Crystals were cryoprotected with mother liquor supplemented with 23% ethylene glycol and flash-frozen in liquid nitrogen. X-ray data sets were collected at 100 K at beamline X06SA of the Swiss Light Source, Villigen, Switzerland. The diffraction data were integrated with XDS [70 (link)] and scaled with AIMLESS [71 (link)], which is part of the CCP4 program suite [72 (link)]. The structure was solved by molecular replacement with PHASER [73 (link)] using a homology model based on PDB entry 2XWR as a search model (generated using SWISS-MODEL [74 (link)]). The structure was then refined using iterative cycles of manual model building in COOT [75 (link)] and refinement in PHENIX [76 (link)]. A summary of the data collection and refinement statistics is given in Supplementary Table S1. Structural figures were prepared using PyMOL (www.pymol.org).