Triiodide derivatization and combinatorial counter-ion replacement: two methods for enhancing phasing signal using laboratory Cu Kα X-ray equipment

A series of experiments performed at Cu Kα wavelength on in-house X-ray equipment are presented which investigate two possibilities for enhancing the experimental phasing signal by means of (i) triiodide/iodide soaks using KI/I₂ and (ii) combinations of counter-ions introduced using the short cryosoak method. Triiodide-derivative crystal structures for five test proteins have been refined and reveal that iodine can bind as polyiodide and single iodide ions through hydrophobic and hydrogen-bonding interactions both at the molecular surface and in intramolecular and intermolecular cavities. In three cases, the structures could be automatically determined with autoSHARP using in-house SAD and SIRAS data. The investigation of combinatorial counter-ion replacement using multiple salts with Na⁺ and Cs⁺ as cations and I⁻ and Cl⁻ as anions reveals that, for the case of hen egg-white lysozyme, significant improvement in phasing signal is obtained by the combined use of salts compared with SIRAS methods using native and single short-soak derivative data sets.