SAD-Phasing of Zn-Substituted Pseudoazurin and the Influence of Data Multiplicity

Denitrifying bacteria contain a blue-copper protein - pseudoazurin - in their periplasm. This protein plays a role in the electron transport chain, as it reduces nitrite to nitric oxide. In order to meet the needs of different spectroscopy experiments, the original copper ion [Cu(II)] has been successfully replaced by other transition metal ions. The native copper ions (Cu2+) of pseudoazurin have been substituted by zinc ions (Zn2+) in order to discover the structure of its crystal using the Zn/S SAD phasing at a wavelength of 2.2Å. For this to be possible, datasets of single wavelength Anomalous dispersion (SAD) are collected initially using high multiplicity. Studies were undertaken to estimate the lowest multiplicity that may be required to get a minute Zn/S substructure for the determination of structures.

Data Collection

A Bruker D8 VENTURE diffraction system was used to collect data from a cryoprotected, flash-cooled single Zn(II)-pseudoazurin crystal at 100 K (Figure 1). The D8 VENTURE has a microfocus IµS sealed-tube equipped with optics HELIOS MX, a detector PHOTON 100, a KAPPA goniostat as well as a low temperature device. An 87-fold multiplicity and 75 runs data set were measured in 4 days devoid of any damage due to radiation.

Zn-pseudoazurin crystal

Figure 1. Zn-pseudoazurin crystal

Data Processing

The reduction and collection of data was completed using PROTEUM2 software. SADABS and SAINT were used to scale and integrate the data. Table 1 displays the collection of data as well as the processing statistics.

Table 1. Data collection and processing statistics

Data collection and processing statistics
Resolution (Å) 2.2
Exposure time (sec) 20
Rotation angle (°) 0.8
Degrees collected (°) 8297
Multiplicity 87.2
Completeness (%) 95.6
Rpim (%) 2.48
I/σs(I) 15.9
Ranom* (%) 3.05

*The average anomalous intensity difference.

Effect of Multiplicity of Data Set on SAD Phasing

The sequential reduction of multiplicity was achieved by omitting the increasing run numbers that were used in the integration of data, phasing, and scaling. Searches for the anomalous atom in all trials were tuned to isolate ten XM (SHELXD) peaks. These results are shown in Table 2. On the basis of the analysis it can be seen that only a tiny fraction is required to determine the substructure and subsequent protein structure phasing. The datasets, which are inclusive of 6 runs and more, displayed the highest peaks as the correct sites of the atom.

Table 2. Success of substructure detecting as a function of multiplicity

Runs Multiplicity Reflections measured/unique* Substructure peaks at atom sites out of ten total
75 87.2 1184616/13583 Zn , 5 Met
44 65 869876/13379 Zn , 5 Met
35 52.9 707379/13378 Zn , 5 Met
29 41.5 555356/13378 Zn , 5 Met
25 36.8 492779/13376 Zn , 5 Met
15 20.3 271355/13343 Zn , 5 Met
7 10.5 139476/13326 Zn , 5 Met
6 8.4 111922/13325 Zn , 4 Met
5 6.6 87693/13289 Zn , 3 Met
4 4.6 61076/13257 Zn , 3 Met

*unmerged anomalous data

Determination of the Structure and Refinement

After the substructure determination was complete, phase extension and density modification were performed using XE (SHELXE) against a high resolution data set of 1.6Å acquired from the same crystal. During the cycles of initial phasing, the structure’s residues, along with anomalous scatterers were traced, while in SAD-phased advanced electron-density maps, SHELX located 96 residues out of a total of 123 residues. Known structures of pseudoazurin were not used in locating the chain. Restrained reiterated refinement using REFMAC54 in addition to manual intervention using COOT6 and XFIT5 showed the map of electron density as illustrated in Figure 2.

Electron density map after model refinement contoured at 2s (pdb accession code 4rh4).

Figure 2. Electron density map after model refinement contoured at 2s (pdb accession code 4rh4).

Conclusion

The D8 VENTURE, with its data processing software PROTEUM2, provided precise and accurate intensities. This was sufficient to obtain phases from anomalous weak scattering of zinc (Zn) and sulphur (S) atoms, derived from data having multiplicity that are generally considered to be too low for effective SAD phasing.

This information has been sourced, reviewed and adapted from materials provided by Bruker AXS Inc.

For more information on this source, please visit Bruker AXS Inc.

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