Abstract
X-ray crystallography is the main source of atomistic information on the structure of proteins. Normal crystal structures are obtained as a compromise between the X-ray scattering data and a set of empirical restraints that ensure chemically reasonable bond lengths and angles. However, such restraints are not always available or accurate for nonstandard parts of the structure, for example substrates, inhibitors and metal sites. The method of quantum refinement, in which these empirical restraints are replaced by quantum-mechanical (QM) calculations, has previously been suggested for small but interesting parts of the protein. Here, this approach is extended to allow for multiple conformations in the QM region by performing separate QM calculations for each conformation. This approach is shown to work properly and leads to improved structures in terms ofelectron-density maps and real-space difference density Z-scores. It is also shown that the quality of the structures can be gauged using QM strain energies. The approach, called ComQumX-2QM, is applied to the P-cluster in two different crystal structures of the enzyme nitrogenase, i.e. an Fe8S7Cys6 cluster, used for electron transfer. One structure is at a very high resolution (1.0 Å) and shows a mixture of two different oxidation states, the fully reduced PN state (Fe82+, 20%) and the doubly oxidized P2+ state (80%). In the original crystal structure the coordinates differed for only two iron ions, but here it is shown that the two states also show differences in other atoms of up to 0.7 Å. The second structure is at a more modest resolution, 2.1 Å, and was originally suggested to show only the one-electron oxidized state, P1+. Here, it is shown that it is rather a 50/50% mixture of the P1+ and P2+ states and that many of the Fe-Fe and Fe-S distances in the original structure were quite inaccurate (by up to 0.8 Å). This shows that the new ComQumX-2QM approach can be used to sort out what is actually seen in crystal structures with dual conformations and to give locally improved coordinates.
Highlights
X-ray crystallography is currently the prime method for obtaining atomic-resolution structural information on biological macromolecules
In terms of computational chemistry, this is a molecular-mechanics potential, in crystallography it is normally based on a statistical analysis of high-resolution crystal structures (Engh & Huber, 1991), rather than on energetic considerations
We have shown that quantum refinement can locally improve crystal structures (Ryde & Nilsson, 2003), decide the protonation state of metal-bound ligands (Nilsson & Ryde, 2004; Cao et al, 2017; Cao, Caldararu & Ryde, 2018; Caldararu et al, 2018) and the oxidation state of metal sites (Rulısek & Ryde, 2006; Cao et al, 2019) and protein ligands (Caldararu et al, 2018), detect the photoreduction of metal ions (Nilsson et al, 2004; Soderhjelm & Ryde, 2006; Rulısek & Ryde, 2006) and solve scientific problems regarding what is really seen in crystal structures (Soderhjelm & Ryde, 2006; Cao, Caldararu, Rosenzweig et al, 2018; Nilsson et al, 2004)
Summary
X-ray crystallography is currently the prime method for obtaining atomic-resolution structural information on biological macromolecules. Such information has been crucial for our understanding of the function of these molecules, opening up the rational construction of enzymes with new functions and the design of new drugs. The resolution of the structures is typically limited, meaning that the exact positions of the atoms are not precisely defined, so that bond lengths and angles, if freely refined, may become somewhat strange. In terms of computational chemistry, this is a molecular-mechanics potential, in crystallography it is normally based on a statistical analysis of high-resolution crystal structures (Engh & Huber, 1991), rather than on energetic considerations
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