The Huck Institutes of the Life Sciences

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans

Kumar AK, Yennawar NH, Yennawar HP, and Ferry JG (2011) Acta Crystallogr Sect F Struct Biol Cryst Commun. 67(7): 775-8


The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Å resolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P213, with unit-cell parameters a = b = c = 92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (VM) of 3.55 Å3 Da-1, corresponding to a solvent content of 65%.

Kumar et al. 2011 Figure 2
A representative crystal of MA1659 protein

Materials and Methods


For crystallization, the purified His-tagged MA1659 protein was concentrated to 20 mg ml-1 in 20 mM HEPES buffer pH 7.5. The protein concentration was estimated using the bicinchoninic acid assay method (Wiechelman et al., 1988). Initial crystallization drops were set up with sitting-drop vapour-diffusion geometry using a Phoenix robot (Art Robbins Instruments, USA). The commercial screens tested for crystallization were PEG/Ion, Index HT, Crystal Screen, SaltRX HT and Additive Screen from Hampton Research and Wizard I and II screens from Emerald BioSystems. Prior to drop setup, the protein solution was incubated with 10 mM NAD(P)H and 1 mM ferrous ammonium sulfate for 24 h at 277 K. This incubation step was crucial to obtain crystals. Each robotic crystallization trial drop consisted of 0.3 µl protein solution mixed with an equal volume of precipitant solution. The plates were incubated in constant-temperature chambers at 294 K. Small brown-coloured crystals (indicative of the presence of Fe) grew within three weeks of setup in condition Nos. 4 and 5 of Index screen. These crystals were reproduced and optimized manually. Larger drops consisting of 4 µl protein solution and 4 µl precipitant solution were set up with a reservoir volume of 0.5 ml. The best condition for the growth of crystals was condition No. 5 of Index screen: 0.1 M HEPES buffer pH 7.5, 2 M ammonium sulfate. Crystal soaking times and cryoconditions were optimized to give the best X-ray diffraction.

Kumar et al. 2011 Figure 3
A representative X-ray diffraction image from the MA1659 protein crystal

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