Critical initial real-space refinement in the structure determination of arginine kinase

Arginine kinase (AK), catalyses the reversible transfer of a phosphoryl gro up between a guanidino phosphate and ADP The family of phosphagen kinases e luded structure determination for over 25 years until an inactive form crea tine kinase (CK) structure was determined [Fritz-Wolf et al. (1996). Nature (London), 381, 341-345]. The structure determination of the active-form tr ansition-state complex was non-trivial, owing to the distant relatedness an d domain reorientation of AK compared with CK, Phases from a molecular-repl acement solution of the large domain, supplemented by single isomorphous re placement and inter-crystal averaging, did not reveal interpretable electro n density for the small domain. Reciprocal-space refinement of the initial model (R-free = 0.54) by any of the commonly used methods, including post f acto application of maximum-likelihood methods, led to overfitting without significant improvement of the partial initial model. By contrast, in the l ocal real-space refinements which proved successful, the interdependence of atoms is limited to immediate neighbors, and atomic positions are not infl uenced by errors or omissions in remote parts of the structure, Modest impr ovement was possible without overfitting, and this was critical to the calc ulation of improved phases. Phases were refined and extended from 4.0 to 2. 5 Angstrom resolution by Fourier inversion of omit maps, combination with i somorphous replacement phases and averaging between crystal forms, after se veral batches of real- and reciprocal-space atomic refinement. The final st ructure refinement, against a 1.86 Angstrom cryo data set yielded a high-qu ality model with R = 0.196 and R-free = 0.224.