Chapter 3. Biochemical and functional characterization of catalytic
4.2 Materials and Methods
4.2.2 Cloning, crystallization and structural analysis of CtXyn30A
The complete methodology of cloning, expression and purification of CtXyn30A has been described in details in the Chapter 2. The additional exercises deployed to obtain highly purified and concentrated protein for seeding crystal drops are as follows:
The recombinant CtXyn30A containing a His6 tag was purified from the cell- free extract by immobilized metal-ion affinity chromatography (IMAC) using a Sepharose column (HiTrap Chelating, GE Healthcare) and was eluted using 50 mM
HEPES buffer (pH 7.5) containing 300 mM imidazole, 1 M NaCl, 3 mM CaCl2.2H2O.
Purified CtXyn30A was buffer-exchanged into 50 mM HEPES buffer (pH 7.5) containing 200 mM NaCl, 5 mM CaCl2using a PD-10 column and was then subjected to gel filtration using a HiLoad 16/60 Superdex 75 column (GE Healthcare) at a flow rate of 1 ml/min. Purified CtXyn30A was concentrated using a 10 kDa cutoff centrifugal concentrator (Millipore, USA) and washed three times with 0.5 mM CaCl2. The purity and molecular mass of the recombinant protein was analysed by 13% (w/v) SDS–PAGE (Laemmli, 1970). The protein concentration was estimated using a molar extinction coefficient (ε) of 91853 per M/cm with a NanoDrop 2000c spectrophotometer (Thermo Scientific).
4.2.2.2 Crystallization conditions for CtXyn30A
The crystallization conditions were screened by the sitting-drop vapour- diffusion method using the commercial screens Crystal Screen, Crystal Screen 2 and PEG/Ion from Hampton Research (California, USA), JCSG from Molecular Dimensions (UK) and an in-house-prepared sparse-matrix screen of 80 conditions using a Nano drop robotic dispensing system Oryx8 (Douglas Instruments, UK).
Drops consisting of 0.9 ml CtXyn30A solution at either 16 or 32 mg/ml and 0.9 ml reservoir solution were prepared at 292 K (Verma et al., 2013). The crystals were harvetsted using harvesting buffer comprise the crystallization buffer containing 5%
higher concentration of the precipitant. The crystals were cryocooled in liquid nitrogen after soaking in the appropriate cryoprotectant (30%, v/v glycerol added to the crystallization buffer for a few seconds, except for the crystal grown in 0.2 M potassium sulfate, 20%(w/v) PEG 3350, which was cryo-cooled in Paratone.
4.2.2.3 Data collection, structure determination and refinement of CtXyn30A
CtXyn30A was crystallized in several conditions and data from all the different crystals obtained were collected on beamline ID29 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) (de Sanctis et al., 2012), Diamond Light Source (Harwell, UK) and at PROXIMA-1 at SOLEIL (Orsay, France) using a PILATUS 6M detector with the crystals cooled to 100 K using a Cryostream (Oxford Cryosystems) (Verma et al., 2013). In addition, several CtXyn30A crystals, crystallized in different conditions, were soaked with xylohexaose or cellohexaose for 1 h, 2 h and 12 h. The data collected from the ESRF, Diamond and SOLEIL were processed using the programs iMOSFLM (Battye et al., 2011) and AIMLESS (Evans, 2011) from the CCP4 suite (Winn et al., 2011) or XDS (Kabsch, 2010). The statistics of data collection are given in Table 4.3.2. All of the crystals tested belonged to space group P1 with one molecule in the asymmetric unit and a solvent content ranging from 45 to 47% and had similar data-collection statistics, despite being grown in different conditions and having different crystal morphologies. The data collected at SOLEIL were from the best diffracting crystal (Fig. 4.3.8D) and were used to solve the CtXyn30A structure. 470° of data were collected with a ∆φ of 0.2°. The crystal was broken/multiple and a second low- resolution data set was needed to index and to find the correct lattice before integrating the high-resolution data with XDS. The resolution was cut at a conservative 1.4 Å (the CC1/2 value is below 0.5 for higher resolution data). The slightly high Rmerge value at this resolution is due to radiation damage from a longer exposure for a P1 data collection. The Matthews coefficient of 2.33Å-3 Da-1 (Matthews, 1968) indicated the presence of one molecule in the asymmetric unit with
a solvent content of 47.2%. The program Phaser (McCoy et al., 2007) was used to solve CtXyn30A structure by molecular replacement approach using, the crystal structure of XynC from Bacillus subtilis 168 (PDB id: 3GTN) as search model (St John et al., 2009) with a final solution giving LLG score of 181 and a TFZ of 24.5 as described in Verma et al. 2013. The structure was further refined and the refinement statistics are shown in Table 4.3.3. A model comprising 1 chain with 389 residues (native protein plus 3 residues in the N-terminal due to the cloning construct) was built from the initial map with program COOT (Emsley et al., 2010). Water molecules were added by visual inspection of the electron density maps, according to hydrogen bond criteria and as indicated by mFo-DFc maps to a total of 647 water molecules, and refined with REFMAC5 (Murshudov at el., 2011), as deemed appropriate from the r behaviour of the cross-validation (Rfree) subset of reflections (5%). The final round of refinement was performed using the TLS/restrained refinement procedure using 3 segments (His-3 to Gln10, Val11 to Pro295 and Gly296 to Val386) as determined by the TLSMD server (Painter and Merritt, 2006), which gave final R and Rfree factors of 0.148 and 0.172, respectively. The final model was verified with PROCHECK (Laskowski, et al., 1993) and checked and validated during submission to the PDB. The r.m.s. deviations of the bond lengths was 0.020 Å and bond angles 1.837 degrees. This structure of CtXyn30A (PDB id: 4CKQ) crystal figure 4.3.8D was used as a model for molecular replacement, to determine other CtXyn30A structures crystallized in different conditions (Fig. 4.3.9). The data were processed using XDS (Kabsch, 2010) and scaled with AIMLESS (Evans, 2011) from CCP4 suite (Winn et al., 2011). All data-collection statistics are indicated in Table 4.3.2.
The three-dimensional structures were solved directly using the program Phaser
(McCoy et al., 2007) and refinement was carried out with REFMAC5 (Vagin et al., 2004) or using Phaser and AUTOBUILD (Terwilliger et al., 2008). The final round of refinement for structures with PDB codes 4uqe, 4uqd, 4uqc and 4uqb was performed in the PDBredo web server (Joosten et al., 2012). For the remaining structures, the final round of refinement was performed using TLS/restrained refinement using 3 segments (His-3-GLN10, Val11-Pro295, Gly296-Val386). In all CtXyn30A structures, both C- and N-terminal regions are well defined in the electron density maps as well as all the side chains. The refinement statistics are indicated in Table 4.3. Visualization and analysis of structures, as well as generation of figures, were conducted using both PyMOL (Schrodinger, 2010) and UCSF Chimera (Pettersen et al., 2004).
Data deposition: Coordinates and observed structure factor amplitudes for CtXyn30A structures have been deposited in the Protein Data Bank in Europe (PDBe), www.ebi.ac.uk/pdbe (PDB id code: 4CKQ, 4UQA, 4UQE, 4UQD, 4UQ9, 4UQC and 4UQB).