Application of joint neutron and x-ray refinement to the investigation of the structure of ribonuclease A at 2.0-A resolution.

Abstract

The principal aim in the determination of protein structure by using neutron diffraction is to find the positions of hydrogen atoms. Since protein crystals used in neutron investigations are usually deuterated (in order to decrease the incoherent scattering by hydrogen atoms), it is also possible to identify highly protected main-chain amide groups (10, 24). While in principle it is possible to determine the neutron structure of a protein de novo, by using either isomorphous replacement with isotopes such as 164Dy or anomalous scattering of 113Cd, 149Sm, or 157Gd, such attempts have not progressed beyond the testing of their feasibility (17). All neutron structures completed to date have utilized x-ray models in the initial stages of the investigation in order to provide the starting set of phases. In most cases the x-ray coordinates were not used after this initial step, and the neutron refinement proceeded on its own (2, 7, 11, 14). On the other hand, Wlodawer and Hendrickson (23) proposed a method of refinement in which the structure is jointly refined with the neutron and x-ray data. In this report we will describe our experience with the application of joint refinement to the determination of the structure of ribonuclease A at 2.0-A resolution, and we will discuss the validity of this procedure.