Macromolecular structure refinement by restrained least-squares and interactive graphics as applied to sickling deer type III hemoglobin

A restrained least-squares (RLS) computer program and two interactive graphics (IG) systems have been used in combination to refine the structure of deer type III hemoglobin. By alternating applications of RLS with examinations and corrections of the atomic model superposed on electron density maps (IG), the residual has been reduced from ~0.42 to ~0.25 and the sites of dubious fit between model and map reduced to ~6% of the residues or ~3% of the atoms. It was possible to fit routinely ~4600 atoms to X-ray intensity data sets ranging from less than 6000 (9.0-4.0 Å resolution) to ~21 500 points (6.0-1.98 Å resolution) employing RLS, which uses interatomic distances to retain structural integrity. Convergence was rapid and many shifts greater than 1 Å were recorded. An in-house graphics display allowed the placement of atoms not in the original atomic model and GRIP, a fast-response interactive graphics system, was used to correct any gross conformational misfit of the atomic model to the electron density maps. The man hours needed to run both GRIP and RLS is less than previously reported real-space methods. The strategy of how RLS and IG can be best applied and how the molecular structure changed during refinement are discussed.