1.7 Å structure of the stabilized REI_v mutant T39K. Application of local NCS restraints

Abstract

The X-ray structure of the T39K mutant of the variable domain of a human immunoglobulin kappa light chain has been determined at room temperature to 1.7 A resolution with a conventional R factor of 0. 182. T39K crystallizes in the triclinic space group P1 [a = 35.4 (1), b = 40.1 (1), c = 43.1 (1) A, alpha = 66.9 (1), beta = 85.4 (1), gamma = 73.8 (1) degrees ]. The unit-cell contains two monomers, related by a non-crystallographic twofold axis. The use of a novel type of local non-crystallographic symmetry restraints on related isotropic displacement parameters and 1-4 distances as incorporated in the refinement program SHELXL improves the model and quality of the maps, but local differences between both monomers in areas subject to different packing contacts can still be observed. 12 overall anisotropic scaling parameters were refined. These may have compensated for the difficulties in accurately scaling single rotation axis image plate data from a triclinic crystal, because of the scarcity of common equivalent reflections. The final model has been used to perform a number of tests on anisotropic scaling, non-crystallographic symmetry, anisotropic refinement, determination of standard uncertainties and bulk solvent correction. It is remarkable that removal of the NCS restraints from the final model caused Rfree to increase. These tests clarify the strategies for optimum use of SHELXL for refinement at medium as opposed to atomic resolution.

A short history ofSHELX

“…Although some facilities such as conjugate-gradient refinement (Konnert, 1976), the free R factor (Brunger, 1992) and non-crystallographic symmetry (NCS) restraints (Usó n, Pohl et al, 1999) were added specially for macromolecular refinement, there are also features missing that would be desirable for macromolecules, such as a way of defining peptide and DNA chains, FFT-based structure-factor calculation, maximum-likelihood refinement, torsion-angle restraints, TLS restraints or constraints for anisotropic refinement and a more sophisticated solvent model. Torsionangle restraints were deliberately left out so that backbone and side-chain torsion angles could be used for verification purposes, e.g.…”

The Crystal Structure of Non‐Modified and Bipyridine‐Modified PNA Duplexes

“…The structure determination was completed by iterative cycles of model building and refinement with SHELXL 41. Local NCS symmetry restraints were only used in the initial phases of refinement in order to improved convergence 42. The course of the refinement was carefully monitored by using 5 % of the reflections to calculated free R factors,43 and completed by restrained anisotropic refinement against all data by using a conjugate gradient method as implemented in SHELXL 41.…”

TLSfrom fundamentals to practice

“…The common displacement of the crystal as a whole (lattice vibrations) (12, 14, 15) contributes equally to all atoms, and therefore it is possible to account for this effect directly while performing overall anisotropic scaling of the model with respect to the diffraction data.…”