Phoenix crystallization robot

Harvested cells were resuspended in buffer A (20 mM Tris-HCl, pH 8, 300 mM NaCl, 1 mM DTT) containing 2 mM MgSO₄, 1 µg ml⁻¹ DNaseI and a cOmplete EDTA-free protease inhibitor cocktail tablet (Roche), followed by lysis via sonication and stirring for 30 min at 4 °C with the addition of 1% Triton-X100. The suspension was centrifuged at 18,000 × g for 30 min and the supernatant was loaded onto a nickel chromatography column (GE Healthcare), washed with Buffer A. The target protein was eluted with a linear imidazole gradient (buffer A containing 500 mM imidazole). The protein was dialyzed overnight at 4 °C against buffer C (20 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM DTT) followed by gel filtration with a Superdex 75 column (GE Healthcare). Finally, ShGdmF was concentrated to 20 mg ml^-1, mixed with 3% sucrose, flash frozen in liquid nitrogen, and stored at –80 °C until use. Crystallization conditions were first screened by sitting drop vapor diffusion at 293 K with 0.2 µl protein solution (10 mg ml⁻¹) and 0.2 µl reservoir solutions using a Phoenix crystallization robot (Art Robbins Instruments). These crystals were optimized by manual screening and finally crystals of ShGdmF were obtained in 100 mM Hepes, pH 7.5, 19–29% (w/v) PEG 4000, 50–200 mM sodium acetate, and 150–300 mM lithium sulfate containing 1 µl protein solution and 1 µl reservoir solution using the hanging drop vapor diffusion method. For ligand-bound structures, crystals were either co-crystallized or soaked with 5 mM substrate for 2 h and cryoprotected in reservoir solutions containing 10% ethylene glycol.

The two major SP elution peaks for PKAc were used independently for crystallographic screening (Fig. S9). The two peaks likely correspond to two different species of PKAc: the first eluted species having more autophosphorylation sites than the second. Based on the electron densities we observed for ApoPKAc/AMP-PNP structures (peak 1) versus C2 (peak 2), we presume that the first peak corresponds to a species that is phosphorylated at residues Ser139, Thr197, and Ser338, while the second species is only phosphorylated at residues Thr197 and Ser338.
We obtained crystals in the presence of the Ca_V1.2 Ser1981 peptide (RGFLRSASLGRRASFHL) using both forms of PKAc (Table S1). Peak 1 resulted in the formation of C1 crystal, and peak 2 formed crystal C2. All proteins used for crystallographic screening were concentrated and buffer exchanged with 20 mM bicine pH 8.0, 150 mM ammonium acetate, 4 mM TCEP. The PKAc samples were mixed with synthetic peptide either directly in the lyophilized powder or in a 5 mM stock solution in the same buffer. Final PKAc concentration for C1 crystal form was 300 μM with AMP-PNP, MgCl₂, and Ser1981 peptide at molar ratio of 1:10:17:10. For the peptide cocrystal structure C2 final PKAc concentration was 250 μM in presence of AMP-PNP, MgCl2, and Ser1981 peptide for a molar ratio of 1:10:10:10. We also screened 300 μM PKAc (peak 1) in presence of a shorter Ser1718 peptide DIGPEIRRAISGDL, AMP-PNP, and MgCl₂ at a molar ratio of 1:10:10:10. No peptide was found to be bound and this thus yielded the apoPKAc crystal form. 96-well plate low volume crystallization plates (Hampton Research) were all set up at room temperature using sitting drop method with ratios 1:1 and 1:2 for precipitant to protein, using a Phoenix crystallization robot (Art Robbins Instruments). All crystal plates were immediately stored at 4 °C. For all structures, the best diffracting crystals originated from the 1:2 precipitant to protein ratio and were transferred to a drop supplemented with 25% ethylene glycol as cryo-protectant.
Crystals were harvested and frozen in liquid nitrogen using Hampton or MiTeGen MicroMounts CryoLoops. All diffraction datasets were processed using HKL2000 (HKL Research Inc.). Best diffracting crystals for C1 appeared in condition 64 of ProComplex crystal screen (QIAGEN) with the following formulation: 0.1 M Hepes pH 7.0, and 18% (w/v) PEG 12,000 Da. Crystals for C2 appeared in condition 79 of JCSG + crystal screen (QIAGEN) with the following formulation: 0.1 M succinic acid pH 7.0, and 15% (w/v) PEG 3350 Da. The apoPKAc crystal was obtained in condition 64 of Classics crystal screen (QIAGEN) with the following formulation: 0.1 M Hepes pH 7.5, 10% (w/v) PEG 8000 Da. Datasets for C1 and apoPKAc were collected at Stanford Synchrotron Radiation Light source (beamlines 12-2 and BL9-2, respectively) at a wavelength of 0.979 Å, using a Dectris Pilatus3 6M detector, while data for the C2 crystal form were collected at the Advanced Photon Source (APS, 23 ID D) at a wavelength of 1.033 Å, also equipped with a Dectris Pilatus3 6M detector. All structures were solved via molecular replacement in Phaser (58 (link)), using PKAc derived from PDB 6MM5 as a search model. Restrained and translation, libration, and screw refinement was carried out using PHENIX (56 (link), 57 (link)).

Crystallization of RasGRP1^CEC was carried out initially with sparse matrix screening using a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA), and thin hexagonal rod-shaped crystals were obtained in a single condition. The initial hit was further optimized through additive screening. Crystals used for data collection were grown by hanging drop vapor diffusion (500 μl reservoir volume) by mixing 1 μl of protein (10 mg/ml) with 1 μl of 0.15 M sodium citrate tribasic, 22% PEG 3350 and 1 mM MnCl₂. Crystals appeared at 20°C in 1–2 days and grew to a maximum length of ∼200 μm over 3–5 days. Crystals were cryoprotected in the crystallization solution with 20% glycerol and flash frozen in liquid nitrogen.
RasGRP1^CC (10 mg/ml) was crystallized in 20 mM sodium acetate (pH 3.6), 22% PEG 3350, 100 mM lithium sulfate and 0.4% formamide by mixing 0.2 μl protein with 0.2 μl of well solution. Square plate-like crystals were harvested after 5–7 days and cryoprotected in the crystallization solution with 20% glycerol. Diffraction data for both RasGRP1^CEC and RasGRP1^CC were collected at 100 K on beamline 8.2.2 at the Advanced Light Source, Lawrence Berkeley National Laboratories.
X-ray data were processed with XDS (Kabsch, 2010 (link)), then Pointless and Scala from the CCP4 program suite (Winn et al., 2011 (link)). Refinement was performed with Phenix.refine (Adams et al., 2010 (link)). For RasGRP1^CEC, an initial molecular replacement solution was found using Phaser (McCoy et al., 2007 (link)) with the RasGRF Cdc25 domain and the core of the SOS REM domain. The location of the C1 domain was identified from anomalous data from the two intrinsic Zn²⁺ ions and the proper orientation was defined by incremental rotation about the axis defining the two metal ions and refinement of the resulting structures. The correct sequence register was determined through identification of 14 of the expected 15 selenium sites using X-ray data for the SeMet-substituted protein (the anomalous peak for residue Met 50 was not present). The position of the Cdc25-EF linker was determined from averaged kick omit maps (Praznikar et al., 2009 (link)) generated in Phenix, which aid in removing model bias. The RasGRP1^CC structure was solved by molecular replacement using the APC coiled coil (Day and Alber, 2000 (link)).
The structural model for RasGRP1^CEC spans residues 53–593 and includes the REM, Cdc25, EF and C1 domains. The electron density for portions of the linkers between the REM and Cdc25 domains (residues 186–192) and between the Cdc25 and EF domains (437–448) is poor and therefore these residues have been excluded from the final model. The model for RasGRP1^CC contains two molecules in the asymmetric unit that form the functional unit. Molecule A contains residues 745–793, while molecule B includes residues 745–786.