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[…] The crystals of the dimeric protein (peak B) were grown using hanging-drop vapor diffusion at 4°C by mixing purified protein at 5 mg/ml in 25 mM Tris·Cl, pH 8.5, 10 mM NaCl, 5 mM DTT with an equal volume of the precipitant solution, 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 7.5% PEG 4000. Wedge-shaped crystals grew to approximately 0.1 mm on a side after a period of 2 weeks. These crystals could be frozen by first transferring them, in four sequential steps, to a solution of 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 15% PEG 4000, followed by transfer in six sequential steps to a solution containing 100 mM Tris·Cl, pH 8.5, 200 mM Na·acetate, 15% PEG 4000, 30% glucose. The monomeric protein (peak A) did not yield crystals.Data were collected at the Cornell High Energy Synchrotron Source (CHESS), beamline A1. Anomalous data, when present, were collected by the inverse beam method. Four sets of data contributed to the structure determination, collected from crystals in 5 mM KAu(CN)2, 1 mM dimercuriacetate (DMA), 3 mM Au(NO3)4, and no adduct, respectively (). A heavy-atom partial structure was solved by inspection of KAu(CN)2 anomalous difference Patterson maps, which revealed two heavy binding sites. Heavy-atom positions were then refined using SHARP (), and difference electron density maps were used to locate a single heavy-atom position in the DMA derivative. Individual mercury atoms within the DMA molecule could not be resolved due to high B-values, so the heavy-atom compound was modeled as a point scatterer. Other derivatives were not useful for MIR phasing, due either to a lack of isomorphism or the presence of overlapping heavy-atom sites. Density modification was performed after heavy-atom refinement in SHARP using the default solvent flipping protocol ().The kinase domain was built into the density modified maps with reference to the already determined Btk kinase domain structure (PDB ID 1K2P), after separately placing the large and small lobes. The SH3 domain was built by comparison with the c-Src SH3 domain (PDB ID 2SRC).We generated a homology model for the SH2 domain from the SH2 domains present in PDB entries for Src, Hck and Abl containing SH3-SH2-kinase constructs (PDB IDs: 1FMK, 1KSW, 2SRC, 2PTK, 1AD5, 1QCF, 2HCK, 1OPK). This model was placed by hand, aligning helices αA and αB into the unambiguous density, then refined as a rigid-body using a 4.0 Å resolution cutoff. Multiple crystal averaging across four distinct data sets (KAu(CN)2, DMA, Au(NO3)4 and native, using the program DMMULTI (; ), revealed the domain swap in the secondary β-sheet portion of the SH2 domain.The structure was refined using alternating cycles of manual rebuilding in the program O () and torsional simulated annealing and grouped B-factor refinement against data to 2.8 Å using the program CNS_SOLVE (; ). A final refinement of the model was performed using PHENIX () to incorporate TLS as three groups, defined as residues 214–260, 261–391, and 392–657. These groups were identified by the automated TLS group search procedure and agreed well with the character of the experimental maps. In all refinement runs in which the SH2 domain was present, an initial temperature factor was refined for the entire SH2 domain, and then the SH2 domain was fixed. SH2 domain geometry is thus strictly a result of the geometry of the initial homology model. Data collection and refinement statistics are summarized in . Illustrations were made in Pymol (DeLano Scientific, New York NY). [...] The bovine PH-TH-kinase construct (residues 1 to 170 connected to residues 384 to 659) was thawed on ice and mixed with twofold excess IP3 (Avanti lipid) and inhibitor CGI1746 (Selleck Inc, Houston TX), and was incubated at 4°C for 30 min before setting up trays. IP3 binds to the protein, but does not activate it. Although present in the crystallization mix, it is not visible in the final electron density (see below). Needle clusters of crystals were obtained by sitting drop vapor diffusion by mixing equal volume of protein solutions at 15 mg/ml with reservoir solution containing 200 mM NaCl, 50 mM MgCl2, and 20% PEG3350. To prepare seeds for microseeding, needle clusters were harvested and combined from 20 drops and transferred into a 1.5-ml eppendorf tube.The bovine PH-TH-kinase construct with activation loop mutations () was prepared in the same way as was the wild-type protein. Equal volumes of protein solution and the reservoir solution containing 200 mM NaCl, 50 mM MgCl2, and 20% PEG3350 were mixed and were equilibrated in a hanging drop tray for 24 hr at 4°C. Microseeding was then performed by striping the wild-type crystal seeds using a cat whisker into the equilibrated drops. Large rock-like crystal clusters appeared ∼4 days after micro-seeding, and single crystals (typical dimensions of 0.20 mm × 0.15 mm × 0.25 mm) can be isolated. For cryo-protection, crystals were soaked in reservoir solution supplemented with 25% glycerol, and flash-frozen in liquid nitrogen and stored at 100 K.Data were collected at the Lawrence Berkeley National Laboratory, Advanced Light Source (ALS), beamline 8.2.1. Data reduction was carried out with the software package HKL2000 (). The structure was determined by molecular replacement using PHENIX () with two search models. These were the structure of the Btk kinase domain that has mutations in the activation loop (see below) and the structure of the PH-TH module. Refinement in PHENIX and COOT () yielded the final models. No density for IP3 is seen in difference electron density maps, even at low contour levels (2σ above the mean in σ weighted |Fo| − |Fc| difference maps). Data collection and refinement statistics are summarized in . Illustrations were made in Pymol (DeLano Scientific). [...] Molecular dynamics trajectories were generated using the Gromacs 4.6.2 package () using the ff99SB-ILDN force field (). All simulations were in water, using the TIP3P water model; appropriate counterions (Na+ and Cl−) were added to neutralize the net charges. After initial energy minimization, the systems were subjected to 100 ps of constant number, volume and temperature (NVT) equilibration, during which the system was heated to 300 K. This was followed by a short equilibration at constant number, pressure, and temperature (NPT, 100 ps). Finally, the production simulations were performed under NPT conditions, with v-rescale thermostats in Gromacs 4.6.2, respectively, in the absence of positional restraints. Periodic boundary conditions were imposed, and particle-mesh Ewald summations were used for long-range electrostatics and the van der Waals cut-off is set at 10 Å. A time step of 2 fs was employed and the structures were stored every 2 ps. His 143, Cys 154, Cys 155, and Cys 165, which coordinate Zn2+ in the PH domain were set to be deprotonated. No additional positional restrains are applied to the Zn2+ ion, and its sp3 tetrahedral coordination remains intact during all MD simulations.The visual molecular dynamics (VMD) analysis toolkit () was used to make some of the root mean square deviation (RMSD) measurements, others were done using PyMol. [...] The active conformation of the Btk kinase domain was modeled based on that of the Lck kinase domain (PDB: 3LCK) (), using Modeller (). The sequence of the kinase domains of Lck and Btk were aligned by ClustalW (). We chose the model with the lowest Discrete Optimized Protein Energy (DOPE: −0.84) as representative of the active conformation of Btk. […]

Pipeline specifications

Software tools CNS, PHENIX, PyMOL, Coot, GROMACS, VMD, MODELLER, Clustal W
Application Protein structure analysis
Chemicals Phytic Acid