Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate.

Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscVC was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers.

Enhancement of tomogram interpretability using the locked self-rotation function.

The interpretation of cryo-electron tomograms of macromolecular complexes can be difficult because of the large amount of noise and because of the missing wedge effect. Here it is shown how the presence of rotational symmetry in a sample can be utilized to enhance the quality of a tomographic analysis. The orientation of symmetry axes in a sub-tomogram can be determined using a locked self-rotation function. Given this knowledge, the sub-tomogram density can then be averaged to improve its interpretability. Sub-tomograms of the icosahedral bacteriophage phiX174 are used to demonstrate the procedure.

Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin.

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450kDa subunits, resulting in a molecular mass >3.3MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form.

Crystallization and preliminary X-ray diffraction analysis of YhbJ from Escherichia coli, a key protein involved in the GlmYZ sRNA regulatory cascade.

The protein YhbJ from Escherichia coli was previously reported to be involved in the regulation of glucosamine-6-phosphate synthase (GlmS) synthesis. YhbJ controls a regulatory cascade composed of the two small RNAs GlmY and GlmZ, which in turn regulate GlmS synthesis. For structural characterization, YhbJ was cloned, expressed and purified to homogeneity by Strep-tag affinity chromatography and size-exclusion chromatography. Multi-angle laser light-scattering analysis revealed its homotrimeric state in solution. The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 91.62, c = 352.82 Å for space group P321 and a = b = 92.72, c = 156.75 Å for one of the enantiomorphic space groups P3(1) or P3(2). Preliminary analysis of the diffraction data suggests the presence of approximately three to seven molecules per asymmetric unit. Owing to the lack of a suitable homologous model, structure determination by means of MIR and MAD methods is required.