Pact suite

Purified SpCBM was concentrated to 33 mg/ml based on the results of precrystallization assay kit screening (Hampton Research). All the subsequent crystallization experiments were done at 20 °C by the sitting-drop, vapour-diffusion method. Initially, commercial kits Crystal Screen, SaltRx, Index (Hampton Research), Wizard, Cryo I&II (Emerald BioSystems), JCSG Suite, PACT Suite and PEGs Suite (Qiagen) were screened by a Honeybee 963 robot system (Genomic Solutions) for protein crystallization. Conditions with crystalline materials were selected for crystallization optimization. After several rounds of optimizations, the best crystals were obtained in 120 mM MMT (molar ratios 1:2:2 of DL-malic acid: MES: Tris Base) buffer pH9.0, 25 % (w/v) PEG1500. Crystals appeared the next day and reached their maximum size within two weeks. Structures of complexes with Neu5Ac derivatives were obtained by co-crystallization with SpCBM. Protein solution (33 mg/ml) containing 5 mM ligand was incubated at 4 °C for 30 min followed by mixing with an equal volume of reservoir solution (100 mM MMT pH9.0 and 28 % (w/v) PEG1500). The crystals appeared the next day and reached maximum size in 3–4 days.

A CAF-1 p150 synthetic peptide with the sequence IKAEKAEITRFFQKPKTPQA was synthesized at the University of Cambridge in-house protein and nucleic acid facility (PNAC). For use in crystallization trials, the synthetic peptide was dissolved in demineralized water to a concentration of 23 mM. Initial crystallization conditions were further refined and optimized with the help of the PACT suite (Qiagen) and Additive Screen (Hampton Research), which contain multiple reagent libraries that enhance the solubility and crystallization of biological macromolecules. Crystal yield was greatly improved under these conditions, and we obtained crystals that exhibited flat surfaces and sharp edges indicating regular lattice plane formation. Well conditions were 0.1M MMT buffer pH 5, 22% (w/v) PEG 1500, and 1.0 M Guanidine hydrochloride. These crystals were subsequently used for diffraction studies. The diffraction data was collected by Drs. Tomasso Moschetti and Andrew Thompson at the Proxima 1 beamline of the SOLEIL synchrotron (Saint-Aubin, France).

Prior to crystallization trials, VLRC was concentrated to a final concentration of 10 mg ml⁻¹ in a buffer containing 10 mM Tris-HCl (pH 8.0) and 50 mM NaCl. Concentration was carried out using a Millipore centrifugal filter device (Amicon Ultra-4, 10 kDa cutoff; Millipore, USA). Screening for crystallization was performed using Wizard Screens I and II (Emerald Biosciences, USA), JBScreen Classic 1–10 (Jena Bioscience, Germany), JCSG + suite, PACT suite, and the PEGs and PEGs II suites (Qiagen, Germany) by the sitting-drop vapor diffusion method in 96-well plates (SWISSCI MRC 2 Well, Jena Bioscience, Germany). A drop of 0.1 µl of the sample was mixed with an equal volume of reservoir solution. The mixture was equilibrated against 0.1 ml of reservoir solution at 293 K. Crystals were grown from PEGs suite #89 (0.2 M potassium phosphate, 20% (w/v) PEG3350). Based on this result, an extensive optimization in a 96-well sitting-drop format was carried out. A diffraction-quality crystal was obtained by mixing 0.2 µl protein solution (15 mg ml⁻¹) and 0.2 µl reservoir solution [0.2 M potassium phosphate, 20% (w/v) PEG3350], and then equilibrating the drops against 60 µl of the reservoir solution at 293 K.