Synchrotron X-ray powder diffraction study of hexagonal turkey egg-white lysozyme

Abstract

The structure of turkey egg-white lysozyme (TEWL) has been refined from high-resolution X-ray powder diffraction data. The sample was rapidly obtained as a polycrystalline precipitate at high protein concentration using 0.5 M NaCl solvent pH 6 and was deposited in the PDB with code 1xft. The diffraction data were collected at room temperature. Molecular replacement was shown to give a suitable starting point for refinement, illustrating that powder data can be sufficient for this approach. Crystallographic models were then refined by combined Rietveld and stereochemical restraint analysis of the powder data (d(min) = 3.35 A), resulting in the extraction of reliable lattice parameters and the refinement of the molecular conformation at room temperature. The structure is hexagonal [space group P6(1)22, unit-cell parameters a = 71.0862 (3), c = 85.0276 (5) A] with 12 symmetry-related molecules in the unit cell, in agreement with previous studies. The results of our analysis are indicative of specific amino acids being disordered at this temperature. Upon cooling, a sudden drop in the lattice parameters at approximately 250 K is observed concurrently with the freezing of the mother liquor. The observation of severe peak broadening below this temperature indicates strain effects accompanying the freezing transition, which are found to be reversible. Finally, a correlation between the unit-cell parameters and the pH of the buffer solution is evident, in a similar manner to earlier observations on HEWL.

Protein powder diffraction – pH variation studies of insulin

“…[1] We have developed a new search/match algorithm which detects and evaluates the anisotropic lattice distortion and intensity deformation due to the preferred orientations. In Figure 1, overlay of the observed powder pattern downloaded from the Search-Match Round Robin-2002 web site [1] and the pattern registered in the ICDD database [2] is shown. The inconsistency between these patterns is due to the lattice distortion of the observed pattern with its lattice constants (a, c)=(9.09, 13.47) from the ICDD pattern (a, c)=(9.19, 13.40).…”

“…In Figure 2, the modified pattern is shown and it agrees well with the experimental pattern. Modern developments of the powder diffraction technique have allowed the investigations of systems with large unit cells like proteins [1][2][3]. One of the main advantages of powder diffraction is the flexibility in crystallisation conditions, as well as being able to observe phase transitions and multiple phases in situ.…”

“…The second method has developed for simultaneous QPA of group of powder patterns with identical qualitative phase composition of samples. The method is development of J.Rius [1] and K.Zangalis [2] methods but differ by iterative refinement mass absorption coefficients of samples and calibrate constants or corundum numbers of phases which made by least squares or simplex methods.…”

A new approach for structure analysis of two‐dimensional membrane protein crystals using X‐ray powder diffraction data

“…According to more recent studies, the synchrotron X‐ray powder diffraction technique has been successfully adapted to various proteins, using state of the art instrumentation and algorithms. Recent progress in powder diffraction data collection11 and interpretation9 has facilitated the study of several biologically interesting proteins 12–18. The successful solution of different protein structures has been achieved via traditional methods in single‐crystal crystallography, such as molecular replacement (MR),11, 13, 17 single isomorphous replacement revealing the molecular envelope,14 and multiple isomorphous replacement showing secondary structure elements of a model protein 15…”

Protein powder diffraction at cryocooled conditions

“…Because of its dietary and pharmaceutical usefulness, various characteristics of aspartame, including the model of its receptor site and its conformation in aqueous solution, have been studied extensively [1,2]. Aspartame is known to grow in different pseudo-polymorphic forms, IA, IB, IIA and IIB, each containing a different amount of water and all having a needle-like morphology.…”