Journal:Acta Cryst F:S2053230X19004151

Phasing with calcium at home

Shuaiqi Guo, Robert Campbell, Peter L. Davies and John S. Allingham ^[1]

Molecular Tour
With better tools for data processing and synchrotron beamlines capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the "first-choice" method for de novo protein structure determination. However, for many proteins, native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5-megadalton calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium-SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure was determined. Given that calcium atoms will be present in a significant percentage of the proteins that remain uncharacterized, many aspects of the data collection and processing methods described here could be broadly applied for routine de novo structure elucidation.

The crystallization conditions, data collection strategies, and structure determination methods used on five sections that together make up >90% of a 1.5-megadalton ice-binding adhesin protein, MpIBP, were described. This enormous adhesin is expressed on the exterior surface of the Gram-negative marine bacterium Marinomonas primoryensis, where it contributes to the bacterium’s ability to bind surface ice in an Antarctic lake for better access to oxygen and nutrients. It is hypothesized that this surface protein uses > 600 Ca2+ to help rigidify the chain formed by one of its domains repeated ~120 times in tandem in region II (RII), and to aid in proper folding and function of domains in the other regions (RI, RIII, RIV, and RV). Here, natural incorporation of Ca2+ within the protein scaffold, as occurs in an abundance of proteins, presented a straightforward path to obtain needed phasing information for de novo protein structure determination.

X-ray crystal structures:

• (PDB 4kdv; cyan). Ca2+ are shown as green spheres.
• (blue).
• (PDB 5k8g; blue).
• (PDB 5j6y; seagreen).
• (PDB 5juh; magenta).

References

1. ↑ Guo S, Campbell R, Davies PL, Allingham JS. Phasing with calcium at home. Acta Crystallogr F Struct Biol Commun. 2019 May 1;75(Pt 5):377-384. doi:, 10.1107/S2053230X19004151. Epub 2019 Apr 26. PMID:31045567 doi:http://dx.doi.org/10.1107/S2053230X19004151