Heinrich Stuhrmann

Small-angle scattering and its interplay with crystallography, contrast variation in SAXS and SANS

Methods of contrast variation are tools that are essential in macromolecular structure research. Anomalous dispersion of X-ray diffraction is widely used in protein crystallography. Recent attempts to extend this method to native resonant labels like sulfur and phosphorus are promising. Substitution of hydrogen isotopes is central to biological applications of neutron scattering. Proton spin polarization considerably enhances an existing contrast prepared by isotopic substitution. Concepts and methods of nuclear magnetic resonance (NMR) become an important ingredient in neutron scattering from dynamically polarized targets.

Contrast variation in X-ray and neutron scattering

This contribution is meant to highlight some progress in those areas of contrast variation which are known to be technically more difficult but which promise interesting applications. These concern the use of the anomalous dispersion of light elements, like sulfur and phosphorus in structural studies and experiments of polarized neutron scattering from nuclear spin polarized samples.

Creating local contrast in small-angle neutron scattering by dynamic nuclear polarization

Low-resolution small-angle neutron scattering measurements can benefit from polarized protons to generate scattering contrast profiles. In a recently developed technique, time-resolved polarized SANS tries to make use of spatial polarization gradients created around paramagnetic centres at the onset of dynamic nuclear polarization. The time constants which describe the build-up of polarization around the paramagnetic centre and the subsequent diffusion of polarization in the solvent were determined by analysing the temporal evolution of the nuclear polarization. The possible use and the limitations of this technique as a spectroscopic tool are discussed.

X-ray spectroscopy and X-ray diffraction at wavelengths near the K-absorption edge of phosphorus

Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.

Neutron scattering from polarised proton domains

Abstract We report on time-resolved small-angle polarised neutron scattering from domains of polarised protons created by dynamic nuclear polarisation in frozen deuterated glycerol–water solutions containing a small amount of paramagnetic centres. In order to observe the rapid build-up of the polarisation of the protons around the paramagnetic ions and to separate it from the much slower polarisation change of the protons in the solvent, we have developed techniques that include stroboscopic SANS and NMR synchronised to cyclic microwave irradiation.

Feasibility and review of anomalous X-ray diffraction at long wavelengths in materials research and protein crystallography

The feasibility and a review of progress in the long-wavelengths anomalous dispersion technique is given in the context of the development of beamline ID1 of the ESRF for such studies. First experiments on this beamline and their analyses are described. The first study reports on the use of uranium which exhibits an unusually strong anomalous dispersion at its M(V) absorption edge (lambda(M(V)) = 3.5 A). The anomalous scattering amplitude of uranium with 110 anomalous electrons exceeds the resonance scattering of other strong anomalous scatterers like that of the rare earth ions by a factor of four. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the MAD method. The anomalous dispersion of a uranium derivative of asparaginyl-tRNA synthetase (hexagonal, a = 124.4 A, c = 123.4 A) has been measured at three wavelengths near the M(V) edge using beamline ID1 of the ESRF. The present set-up allowed the measurement of 10% of the possible reflections at a resolution of 8 A. This is mainly due to the low sensitivity of the CCD camera. The second study, involving DAFS experiments at wavelengths near the K-absorption edge of chlorine (lambda(K) = 4.4 A), reports the use of salt crystals which give rise to much stronger intensities of diffraction peaks than those of protein crystals. In the case of a crystal of pentamethylammonium undecachlorodibismuthate (PMACB, orthorhombic, a = 13.00 A, b = 14.038 A, c = 15.45 A), all reflections within the resolution range from 6.4 A to 3.5 A and the total scan width of 24 degrees were collected. The crystalline structure of PMACB implies two chemically distinct states of the Cl atom. Consequently, different dispersions near the K-edge of chlorine are expected. The dispersion of the intensity of five Bragg peaks of the PMACB crystal has been measured at 30 wavelengths. The relative success of these preliminary experiments with X-rays of long wavelength shows that the measurement of anomalous X-ray diffraction at wavelengths beyond 3 A is feasible. Starting from the experience gained in these experiments, an increased efficiency of the instrument ID1 by two to three orders of magnitude will be achieved in this wavelength range. A comparison with different techniques of anomalous diffraction which rely on the use of argon/ethane-filled multiwire chambers and image plates as detectors for wavelengths near the K-edge of sulfur and phosphorus is also given.

Neutron contrast variation and dynamic nuclear polarisation

Abstract The spin-dependent interaction of neutrons with nuclei is best exploited in experiments of polarised neutron diffraction from dynamic polarised nuclei. NMR methods like nuclear depolarisation or nuclear spin reversal by AFP act selectively on nuclei whose Larmor frequencies differ either because of their magnetic moments or their proximity to paramagnetic centres. The combined use of NMR and EPR methods extends the techniques of polarised neutron scattering to more complex systems.

Localization of proteins and tRNA molecules in the 70S ribosome of the Escherichia coli bacteria with polarized neutron scattering

Isotopic substitution methods are widely used in neutron scattering for the determination of the in situ structure of macromolecular components in quaternary structures. The contrast created by the substitution of the hydrogen isotope l H (proton) by 2H (deuteron) is the most prominent example. A further increase of contrast by a factor of three is possible by polarized neutron scattering from polarized nuclear spins. This offers the possibility of measuring small labels, such as proteins, which contribute less than 0.5% to the whole ribosomal mass, or weakly contrasted molecules, such as tRNA ligands, in the 70S ribosomes. In this study, the positions of the proteins

The electron-spin–nuclear-spin interaction studied by polarized neutron scattering

6 and S10 of the Esherichia coli ribosome with respect to the whole 70S ribosome have been determined by nuclear-spin contrast variation. Furthermore, the localization of two weakly contrasted tRNA molecules bound to the pre- and post-translocational 70S ribosome, respectively, is presented. So far, no other technique has allowed the determination of the in situ structures of these molecules.

Polarized Neutron Scattering from Polarized Nuclei Near Paramagnetic Centers

Dynamic nuclear spin polarization (DNP) is mediated by the dipolar interaction of paramagnetic centres with nuclear spins. This process is most likely to occur near paramagnetic centres at an angle close to 45 degrees with respect to the direction of the external magnetic field. The resulting distribution of polarized nuclear spins leads to an anisotropy of the polarized neutron scattering pattern, even with randomly oriented radical molecules. The corresponding cross section of polarized coherent neutron scattering in terms of a multipole expansion is derived for radical molecules in solution. An application using data of time-resolved polarized neutron scattering from an organic chromium(V) molecule is tested.