Yoshiaki Minezaki

Neutron Laue diffractometry with an imaging plate provides an effective data collection regime for neutron protein crystallography.

Neutron quasi-Laue diffraction data (2 Å resolution) from tetragonal hen egg-white lysozyme were collected in ten days with neutron imaging plates. The data processing Laue software, LAUEGEN, developed for X-ray Laue diffractometry, was adapted for neutron diffractometry with a cylindrical detector. The data analysis software, X-PLOR, was modified and used for the refinement of hydrogen atoms, and the positions of 960 hydrogen atoms in the protein and 157 bound water molecules, were determined. Several examples are given of the methods used to identify hydrogen atoms and water molecules.

Aggregation in supersaturated lysozyme solution studied by time-resolved small angle neutron scattering

Abstract A model of the lysozyme crystallization process has been given by time-resolved small angle neutron scattering from lysozyme in supersaturated solution. In supersaturated solution larger aggregates (Type I), with a radius greater than several hundred angstrom, co-exist with a much larger number of smaller particles (Type II), the radius of which is between 25 and 40 A. The size distribution of Type I is poly-dispersive and that of Type II is monodispersive. As time elapses, the structure of Type I aggregates does not change so much. On the other hand, the radius of Type II aggregates increases with time, but stops at about 14 h, and then decreases. Nucleation occurs at about 14 h.

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions).

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.

SMALL ANGLE NEUTRON SCATTERING FROM LYSOZYME IN UNSATURATED SOLUTIONS, TO CHARACTERIZE THE PRE-CRYSTALLIZATION PROCESS

Abstract The small angle neutron scattering (SANS) method was used to study the crystallization process of lysozyme from the microscopic molecular level. SANS was observed from unsaturated solutions of lysozyme in D 2 O. By adding NaCl, lysozyme molecules start to aggregate, and even when the NaCl concentration is increased to supersaturation, SANS spectra show aggregation in the initial stage. The difference of the aggregation states in the unsaturated or supersaturated for protein crystallization research.

Crystallization of a large single crystal of a B-DNA decamer for a neutron diffraction experiment by the phase-diagram technique

Crystallization of a large single crystal of a B-DNA decamer, d(CCATTAATGG), for a neutron-diffraction experiment has been accomplished by an analysis of its solubility phase diagram and a large single crystal was successfully crystallized at around the minimum solubility point of the oligonucleotide: 30%(v/v) MPD, 100 mM MgCl(2) pD 6.6 using 0.4 ml D(2)O solutions of the DNA (sample concentration 1.5 mM). It is confirmed that the resulting crystal (dimensions: 1.7 x 1.3 x 0.6 mm) diffracts sufficiently well for neutron data collection.

Polar structure of lysozyme aggregates in unsaturated solution determined by small-angle neutron scattering – contrast variation method

Abstract The structure of lysozyme aggregates in unsaturated solution was studied by means of the small-angle neutron scattering – contrast variation method. The fact that lysozyme molecules aggregate even in unsaturated solutions and the size of aggregates increases with the increase of the NaCl concentration has been confirmed. Constituent polar molecules in aggregates align parallel with each other on aggregate formation. This indicates that the aggregates could not become precursors of nucleation since the parallel molecular arrangement is not the motif for the crystal of tetragonal form.

A neutron diffractometer for biological macromolecules using neutron imaging plates

Abstract Neutron imaging plates (NIPs), developed recently, have a good spatial resolution and availability of a large sensitive area. The NIPs having such characteristics may be particularly useful for neutron-diffraction measurements of biological macromolecules. We have constructed a neutron diffractometer dedicated to crystallography of biological macromolecules using the NIP, in the guide hall of the reactor JRR-3M at Japan Atomic Energy Research Institute. Neutrons are monochromatized with an elastically-bent-silicon monochromator ( λ = 0.22 nm). The diffraction patterns are detected with the NIP of 400 × 520 mm 2 at a distance of 150–300 mm from a sample crystal. A sequence of the measurements (exposure, reading the NIP, erasure of the pattern, and starting the next exposure) is done automatically. We measured the diffraction patterns from hen egg-white lysozyme crystals, and obtained the patterns which can be processed to derive integrated intensity of each diffraction spot.

Data acquisition of neutron crystallography on tetragonal and triclinic forms of hen-egg-white (HEW) lysozyme with an elastically bent Si monochromator

Abstract An elastically bent silicon (EBSi) as a monochromator has been optimized for neutron diffractometers of biocrystallography. It was found that several stacked thin Si plates were easier to be bent much for the near focusing point and they increased neutron reflectivity by aligning the plates. Currently, an EBSi(1 1 1) monochromator system was equipped at a diffractometer (BIX-I). It took 50 days to collect about 12 000 reflections of hen-egg-white lysozyme. The minimum d -spacing was 2.1 A.

Neutron Laue diffractometry with an imaging plate provides an effective data collection for neutron protein crystallography

Abstract Neutron Laue diffraction 2 A data from tetragonal hen egg-white (HEW) lysozyme were collected for 10 days with neutron imaging plates. All the 960 hydrogen atoms in the lysozyme and 157 bound water molecules could be identified. The final R-factor is 19.5%. Several interesting features of hydrogen atoms and bound water molecules of HEW-lysozyme were found as follows. (1) The crystal was grown at pH 7.0. There are no hydrogen (deuterium) atoms bound to carboxylate oxygen atoms of Glu35 and Asp52, which explains why lysozyme is less active at pH 7.0. (2) Hydrogen atoms and deuterium atoms are clearly identified. As an example, the omit map around indol ring of Trp111 is shown.