Computational protocol: Mechanistic insights from the crystal structures of a feast/famine regulatory protein from Mycobacterium tuberculosis H37Rv

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Protocol publication

[…] The protein was crystallized using the hanging drop technique by equilibrating protein drops against reservoirs containing 100 mM MES pH 6.0, 0.75 M lithium acetate at 22°C. Heavy atoms were screened by band shift assays on a native gel. Crystals were soaked in appropriate heavy atom solutions for varying times and concentrations. Data were collected using a Rigaku RU300 X-ray generator coupled with MAR345 detector or alternatively with MAR345-DTB detector on crystals mounted in quartz capillaries at room temperature. Data were processed using MOSFLM () implemented in the CCP4 package () and collection parameters are summarized in Supplementary Table 1. While crystals of the apo protein diffract to 2.5 Å resolutions, the crystals of the Phe complex diffract to 2.2 Å.Extensive molecular replacement calculations using available archaeal and bacterial Lrp structures were inconclusive. Phases were therefore calculated by MIRAS methods using the SOLVE/RESOLVE package () using three derivatives; the first one being silver nitrate, (crystals soaked in 80 mM solution for 2 days) and the latter two data sets from potassium tetra-chloro-platinate (2.0 mM, overnight soak) and cadmium iodide (1.7 mM, 1 h soak) derivatives. The solution output by SOLVE had a figure of merit 0.28 before solvent flattening and this went up to 0.68 after density modification and phase extension using RESOLVE. The structure solution parameters are summarized in Supplementary Table 1a. RESOLVE could trace ∼15% of the residues. The map quality was sufficient to trace the remaining chains manually using TURBO–FRODO (). Refinements were carried out using REFMAC () after setting aside ∼10% of the reflections for monitoring the free R-factor. Structures of the complexes were solved using difference Fourier techniques. Omit maps were examined to avoid bias. Quality of the structures was checked using PROCHECK () and the geometric parameters as also the Ramachandran plot () falls into acceptable ranges. [...] Analysis of the inter-subunit contacts and ligand–protein interactions, accessible surface area, etc. were carried out using CONTACTS and other tools implemented in the CCP4 package (). Structural superposition was carried out using ALIGN () and PROFIT ( Figures were generated using Pymol (), CCP4MG () and InsightII ( […]

Pipeline specifications

Software tools CCP4, PROCHECK, PyMOL, CCP4mg
Application Protein structure analysis
Organisms Mycobacterium tuberculosis, Dipturus trachyderma, Mycobacterium tuberculosis H37Rv
Diseases Tuberculosis