Computational protocol: Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis

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Protocol publication

[…] rSALO(P) was buffer exchanged and concentrated to 24 mg/ml in 50 mM Tris HCl pH 8.0 using a 5K MW cutoff centrifugal concentrating device (Millipore). The initial protein concentration was confirmed by measurement of OD280 prior to setting up crystallization experiments. Crystallization conditions were screened using commercial screens from Hampton Research at 298K. Crystals were grown by vapor diffusion in sitting drops, which were equilibrated against well containing 0.5 ml crystallization solution. Drops were prepared by mixing 1.5 μl of protein solution with an equal volume of crystallization solution. No crystals were obtained for protein produced in mammalian cells, possibly because of the presence of the N-terminus vector derived sequence. rSALO(P) crystallized within 16 hours from a precipitant solution containing 0.02M calcium chloride, 30% v/v MPD and 0.1 M sodium acetate pH 4.6. Larger crystals with dimensions 0.8 mm X 0.5 mm X 0.3 mm were obtained within 48 hours by setting up larger drops using a ratio of 4 μl of protein to 1.5 μl of the same precipitant solution.Since crystals grew in solutions that contained adequate cryoprotectant, they were flash-cooled directly in a stream of N2 gas at 113 K prior to collecting diffraction data. X-ray diffraction data were collected at the Baylor College of Medicine core facility (Rigaku HTC detector, Rigaku FR-E+ SuperBright microfocus rotating anode generator, with VariMax HF optics) using the Crystal Clear (d*trek) package []. Data was integrated using MosFLM and scaled with SCALA []. Crystallographic data is shown in . [...] The structure of rSALO(P) was solved using single-wavelength anomalous dispersion with the anomalous signal from sulfur at Cu-Kalpha wavelength. FA values were calculated using the program SHELXC []. Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was set to 1.94 Å. The location of 89 atoms (C, S, N, O) were automatically determined using the program SHELXD [] and based on the results of this automated search 82.08% of the model was built using the program ARP/wARP [, ]. Since the difference between Rfactor and Rfree remained unreasonably high, the structure was subsequently refined in a lower symmetry space group with a dimer in the asymmetric unit. The final model was obtained by iterative manual model building cycles using the program Coot [] followed by structure refinement with REFMAC5 [][] and PHENIX[]. Structural figures were generated using PyMOL []. The refined coordinates and structure factors have been deposited in the RCSB protein databank under accession code 4LU2. […]

Pipeline specifications

Software tools SHELX, iMosflm, CCP4, ARP/wARP, Coot, REFMAC5, PHENIX, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Drosophila melanogaster, Leishmania, Komagataella pastoris, Homo sapiens
Diseases Drug Hypersensitivity, Gastrointestinal Diseases, Infection, Leishmaniasis, Salivary Gland Diseases