Protein crystallization and structure determination

POT1C-TPP1(PBD), crystal screening produced a crystal hit under sitting-drop vapour diffusion at room temperature in a crystallization buffer containing 2.4 M KCl, 50 mM K/Na Tartrate, 20 mM BaCl₂, and 0.1 M Sodium Citrate, pH 5.5. (A longer construct of POT1C consisting of residues 325–634 produced a different crystal form that belonged to the P1 space group and diffracted to 3 Å at best.) The new crystal form belongs to the P4₁22 (sg91, 1 copy in asymmetric unit) and diffracted to 2.1 Å. The native data set (containing Zn) was collected from 2 crystals at 1.03317 Å wavelength at BL12-2 SSRL to 2.1 Å resolution. The radiation damage was slowed with a careful absorbed dose estimate, allowing high-multiplicity and accumulating a significant anomalous signal from the present Zn and Sulfurs in the protein. Both the native and derivative data were processed with XDS (autoxds script at SSRL) with a zero dose correction.

The structure was solved using a single methyl mercury (meHg) derivative using the SAD approach as implemented in SHELXCDE using the graphical interface of the HKL2MAP software. SHELXD identified five well defined Hg sites and SHELXE extended and optimized the initial phases to 2.1 Å and generated a preliminary structure of 330 residues with excellent contrast (0.943) and connectivity (0.843) resulting in FOM of 0.605. Subsequently the model was traced using 10 cycles of BUCCANEER. The resulting model was almost complete—358 sequenced residues. The remaining model was improved in COOT and refined with BUSTER (version 2.10.2). The refinement converged to R/R_free=17.3/19.3% to 2.1 Å resolution. The model has excellent stereochemistry as examined by MOLPROBITY server (http://molprobity.biochem.duke.edu/).