Poster Sessions C732 MS82.P01 Acta Cryst. (2011) A67, C732 High resolution neutron diffraction on a tiny perdeuterated crystal Andre Mitschler, a Matthew Blakeley, b Isabelle Petit-Haertlein, b Michael Haertlein, b Christophe Mueller-Dickmann, c Alexandre Popov, c Alberto Podjarny, a a IGBMC, CNRS, INSERM, UniStrasbourg, Illkirch, (France) b ILL,Grenoble, (France) c ESRF-Grenoble, (France). Perdeuterated Type III Anti-Freeze Protein (AFP-D, M.W.=7kDa) has been overexpressed and crystallized in D2O in the Deuteration Laboratory at the Institut Laue Langevin (ILL) in Grenoble, France. Neutron Laue data collection up to 1.85A was performed at room temperature (RT) on the new LADI-III Diffractometer at ILL using a sub-cubic millimetric crystal volume (0.15mm3). The crystal quality was very good, as shown by the mosaicity (0.05 - 0.10° refned by Program HKL2000). Detailed data processing statistics will be given. The structural identity between the hydrogenated and perdeuterated proteins has been checked by comparing with the structure refned against X-ray diffraction data (RT, ID29, Synchrotron ESRF , 1.05 A resolution). Keywords: neutron diffraction, protein crystallography, perdeuteration MS82.P02 Acta Cryst. (2011) A67, C732 Proton polarization technique for neutron protein crystallography (NPC) N.Niimura, a T.Chatake, b I.Tanaka, a,c K.Kusaka, a a Frontier Research Center for Atomic Sciences, Ibaraki Univ., b Kyoto Univ. Reactor, c Faculty of Engineering, Ibaraki Univ. (Japan). E-mail: niimura@ mx.ibaraki.ac.jp The hydrogen atoms in a protein have a large neutron incoherent- scattering cross section, which makes the background signal large and the hydrogen atoms sometimes diffcult to see. The current method for reducing this incoherent scattering is to deuterate the sample crystal, either partially by soaking it in heavy water solution or completely by culturing the protein-producting bacteria in deuterated media from the beginning. However, there always remains the question of whether or not the deuteration affects the folding structure of the protein, because the living cells that produce the protein never live in nature in a heavy water environment for so long. It might be possible to carry out a neutron protein crystallography experiment with a normal protein crystal, without deuterating it. The method would be to polarize the spins both of the neutrons and of the protons (hydrogen atoms) in the protein. This method, called the proton polarization method (PPM), would enable a very accurate determination of the hydrogen atoms in a protein without deuteration. The effective coherent-scattering length of a hydrogen atom (proton) is given as a function of the polarization. When the proton is not polarized, scattering length of hydrogen atom, bH(0) = -0.375∙10 - 12 cm. When the proton is 100 % polarized and the polarization is parallel with the neutron, bH(++) = 1.085∙10 -12 cm. When the proton is 100 % polarized but the polarization is anti-parallel with the neutron, bH(+-) = -1.835∙10 -12 cm. Thus it is seen that the effective coherent- scattering lengths of hydrogen atoms with parallel and anti-parallel spins, relative to the neutron polarization, are very different from each other. If this nature could be exploited, it would be a unique way to determine the position of the hydrogen atoms in proteins. Firstly, all of the protons (hydrogen atoms) in all of the protein molecules in a single crystal must be polarized. A small amount of paramagnetic centers is added to the sample. In a moderately strong magnetic feld of 2.5 T at temperatures less than 1 K, microwave irradiation will polarize the nuclear spins. Secondly, the neutron beam must be polarized parallel to the protein protons and then used to make a typical diffraction experiment. Thirdly, the neutron polarization must be reversed, and another diffraction experiment should be made. When the difference Fourier map, ||F(++)(hkl)|-|F(+- )(hkl)|, is calculated, only the hydrogen atoms (protons) appear in the difference map. Moreover, since the signs of bH(++) and bH(+-) are opposite, the difference between them becomes much larger. Indeed, the ratio of this difference to the scattering length of unpolarized hydrogen atoms is about eight ((bH(++) - bH(+-) / bH(0)≈ 8), so that the Fourier peak of hydrogen atoms in this method is enhanced eight times over that obtained in normal NPC. The technique of producing polarized neutrons is well established. The polarization of protons in protein molecules in solution has been tried successfully. [1] However, the polarization of protons in protein molecules in a single crystal has never been tried yet. The remaining signifcant hurdles are (1) how to realize the cooling of a protein crystal at 1 mK, and (2) how to dope the crystal with paramagnetic materials, which are essential to initialize the polarization of protons. The frst hurdle might be overcome by the development of the technique of high- pressure cooling of protein crystals. [2] The second hurdle might be overcome by upgrading the technique of polarizing the protons in protein molecules in solution. [1] H. Stuhrmann, et al. (1986), Eur. Biophys. J. 14,1-6. [2] C. Kim,C. et al. (2005), Acta Cryst. D61, 881-890. Keywords: neutron protein crystallography, deuteration, polarized neutron MS82.P03 Acta Cryst. (2011) A67, C732-C733 Overview of the IBARAKI biological crystal diffractometer (iBIX) at J-PARC Ichiro Tanaka, a,b Katsuhiro Kusaka, b Takaaki Hosoya, a,b Kurihara Kazuo, c Takashi Ohhara, c,d Taro Yamada, b Katsuaki Tomoyori, b Takeshi Yokoyama, b Nobuo Niimura, b a College of Engineering, Ibaraki University, Hitachi, Ibaraki (Japan). b Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Tokai, Ibaraki (Japan). c Quantum Beam Science Directorate, (Japan) Atomic Energy Agency (JAEA), Tokai, Ibaraki (Japan). d Research Center for Neutron Science & Technology, Comprehensive Research Organization for Science and Society (CROSS), Tokai, Ibaraki (Japan). E-mail: i.tanaka@mx.ibaraki.ac.jp. The IBARAKI Biological Crystal Diffractometer (iBIX), a new diffractometer for protein crystallography at the next generation neutron source at J-PARC (Japan Proton Accelerator Research Complex), has been constructed and has been operational since December 2008. Preliminary structure analyses of organic crystals and a protein crystal showed that iBIX has high performance even at 120 kW operation [1]. From November 2010, J-PARC proton power has increased up to 220 kW and full data sets of two protein crystals were collected successfully. Now iBIX has 14 detector units, the basic part of data reduction software (STARGazer) and an equipment of cryostream cooler to 20K. According to the performance of iBIX measurements, it turned out that it is possible to collect a full data set of a protein in 3-4 days for a crystal of 1 mm 3 in volume and to reduce data from a crystal whose unit cell dimension is as long as about 200 Angstrom. When J-PARC reaches at 1MW, one can expect nearly a 10 times higher effciency at iBIX than that at present because iBIX detector units will be added up