Table 1.

Crystallographic details of LmTBCC-C.

Data collection
Wavelength (Å)	0.97907
Space group	P21
Cell dimensions
a, b, c (Å)a, b, g (°)	37.6 93.2 48.390.0 108.4 90.0
Resolution (Å)	46.6–2.2 (2.27–2.20)a
Rmerge/<I/sI>	10.2 (23.9)/17.5 (6.8)
CC ½b	0.998 (0.967)
Completeness (%)/multiplicity	98.3 (86.8)/11.9 (7.1)
Anomalous completeness	97.7 (82.5)
Anomalous multiplicity	6.0 (3.4)
Wilson B(Å2)	14.6
Refinement
No. reflections (total/Rfree)	15026 (792)
Rwork/Rfree	15.8/19.7
No. atoms protein/water	2697/300
B-factors(Å2)
Protein (chainA/chain B)	19.8/20.1
Water	28.4
r.m.s. Deviations
Bond lengths (Å)/angles (°)	0.010/1.326
Ramachandran distribution (%)c
Favored/outliers	98.2/0

The gene fragment encoding residues 152–355 of LmTBC-C from Leishmania major strain Friedlin identified in GeneDB (LmjF.36.3160, [21]) was amplified from genomic DNA using PCR. To permit the use of selenomethionine for phase determination a single mutation, Leu223Met was introduced (Quikchange mutagenesis, Stratagene). The gene was cloned into a modified pET15b plasmid to encode an N-terminal His-tag followed by a tobacco etch virus (TEV) protease cleavage site. The resulting vector was transformed into Escherichia coli B834 (DE3), and cells grown in Selenomethionine Medium (Molecular Dimensions, UK), expression induced with 1 mM IPTG at an OD₆₀₀ 0.6 and growth continued at room temperature for 16 hours. Cells were harvested by centrifugation and resuspended in 50 mM Tris–HCl pH 7.5, 250 mM NaBr, 20 mM imidazole before storage at −20 °C.

Thawed cells were lysed using French Press at 16 kpsi and lysate was clarified by centrifugation at 37,500 × g for 30 min at 4 °C. Soluble supernatant was filtered (0.2 μm) and loaded onto a 5 mL HisTrap HP column (GE Healthcare) pre-equilibrated with 50 mM Tris–HCl, 250 mM NaBr pH 7.5 for an initial affinity chromatography capture step. Elution of LmTBCC-C was performed by applying an imidazole gradient with the target protein eluting at approximately 140 mM. The product was treated with TEV protease at 30 °C for 2 h. Dialysis at room temperature, to remove excess imidazole, was followed by reverse affinity chromatography prior to a final purification step with size exclusion chromatography using a calibrated Superdex 200 26/60 gel filtration column and the equilibration buffer. The protein eluted with an estimated mass of 20 kDa, which corresponds to that expected for a monomeric sample (20.4 kDa). The sample was pooled, buffer exchanged into 10 mM Tris–HCl, 100 mM NaBr pH 7.5 and concentrated using a centrifugal concentrator (10 kDa cutoff, Sartorius) prior to crystallization. The protein concentration was determined by measurement of absorbance at 280 nm and an estimated extinction coefficient 38,680 M⁻¹ cm⁻¹[22]. Poor quality crystals were produced at 18 °C by the hanging drop vapor diffusion method using 0.75 μL of protein solution at a concentration of 7 mg mL⁻¹, mixed with 0.75 μL of reservoir containing 100 mM MES (4-morpholineethanesulfonic acid) pH 6.5, 25–30 % PEG 2000 MME (polyethylene glycol monomethyl ether). A crystal was placed into an Eppendorf tube with 100 μL of reservoir and a small nylon ball was added before vortexing for 30 seconds to create a micro-crystal suspension. Fresh conditions were prepared with 100 mM MES pH 6.7, 22% PEG 2000 MME in the reservoir, and protein solution as before but at a reduced concentration of 3 mg mL⁻¹. A cryo-loop was used to streak the micro-seed suspension into the conditions and the plates stored at 18 °C. Well formed needles (40 × 40 × 500 mm) appeared in several days. Single-wavelength anomalous dispersion (SAD) data were measured from a single crystal at −170 °C on beam line I24 of the Diamond Light Source with a Pilatus 6 M detector. A helical data collection protocol to minimize radiation damage was used. Data were indexed and integrated using XDS [23] and scaled using AIMLESS [24]. The structure was solved via SAD-phasing using Phenix AutoSolv [25]. Two molecules of LmTBCC-C constitute the asymmetric unit and each contains two SeMet residues. These four Se positions were identified and provided an initial figure-of-merit 0.44. The density modification step yielded an improved figure-of-merit of 0.69 and was followed by automated model building to produce a partial model consisting of 311 residues giving an R/R_free of 24.2%/29.1% and a map-model correlation coefficient of 0.79. The model was then completed with the graphics software COOT [26]. Refinement was performed in REFMAC5 [27] utilizing Translation/Libration/Screw refinement [28], and alternated with rounds of electron and difference density map inspection and model manipulation together with ligand incorporation using COOT, and the incorporation of waters and alternate conformer side chains. Non-crystallographic symmetry restraints were not employed. MOLPROBITY [29] was used to investigate model geometry in combination with the validation tools provided in COOT.

^aValues in parenthesis are for the highest resolution shell.

^bPearson correlation coefficient [30].

^cCalculated using the Molprobity server (http://molprobity.biochem.duke.edu).