Research Article: Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD

Date Published: November 01, 2016

Publisher: International Union of Crystallography

Author(s): Andrew F. Bent, Greg Mann, Wael E. Houssen, Vitaliy Mykhaylyk, Ramona Duman, Louise Thomas, Marcel Jaspars, Armin Wagner, James H. Naismith.


The first crystal structure of ThcOx, a cyanobactin oxidase, has been determined to 2.65 Å resolution using S-SAD phasing. This is the first structure reported from the purpose-designed long-wavelength beamline I23 at Diamond Light Source.

Partial Text

The majority of structures from macromolecular crystals can nowadays be solved by molecular replacement. However, in the absence of suitable homology models, experimental phasing is the method of choice to overcome the crystallo­graphic phase problem. This method requires measurements of the small anomalous differences arising when tuning the wavelength of the X-rays towards the absorption edges of atoms bound to the structures. The most successful label used is selenium by substituting the amino acid methionine by selenomethionine. However, this technique is not universal as it is not compatible with all expression systems and, even if the labelled protein can be produced, crystallization fails in some cases.

The protein crystals belonged to space group P412121, with two molecules found in the asymmetric unit. Simple phase extension from the long-wavelength data used for phasing was not possible owing to non-isomorphism between the two crystals. The c axis is more than 10% larger in the long-wavelength data, and the unit-cell volume is about 15% larger compared with the data obtained from the crystal which diffracted to the highest resolution. Hence, molecular replacement was needed to phase the high-resolution data. This crystal-to-crystal variability was typical. The structure is composed of a novel domain and an FMN nitroreductase domain. Each molecule in the asymmetric unit consists of 16 α-helices and 17 β-sheets (Fig. 4 ▸; structural figures were created using PyMOL v. from Schrödinger). The final structure contains residues 3–222 and 233–469 in chain A and 1–222 and 231–473 in chain B. In both monomers, the missing residues are located between α-helices 7 and 8 (the linker between the two domains) and at the N- and C-termini. These residues are presumed to be disordered in the structure. There are four cysteine residues per molecule, none of which form disulfide bonds.

We have reported the first structure of an oxidase protein from a cyanobactin pathway. The structure was determined by native S-SAD phasing on the new I23 beamline at Diamond Light Source. A wavelength of 3.10 Å was used to measure a sufficiently large anomalous signal to experimentally phase the structure of ThcOx from a crystal which diffracted to 3.15 Å resolution with useful anomalous data extending to only 4.2 Å resolution. These resolution limits would traditionally be unfavourable for phase determination by native S-SAD, particularly given the large size of the asymmetric unit (a dimer of 954 residues in total) with relatively few S atoms. Furthermore, the inability to extend the phases to a higher resolution data set, caused by non-isomorphism, required that the initial phases at 3.15 Å resolution had to be of sufficient quality to allow model building. Thus, ThcOx represented a particularly challenging test for the I23 beamline and the successful structure determination validates its performance.




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