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The nascent suite of single-crystal neutron diffractometers at the Oak Ridge National Laboratory has no equal at any other neutron scattering facility worldwide and offers the potential to re-assert single-crystal diffraction using neutrons as a significant tool to study nuclear and magnetic structures of small unit cell crystals, nuclear structures of macromolecules, and diffuse scattering. Signature applications and features of single-crystal neutron diffraction are high resolution nuclear structure analysis, magnetic structure and spin density determinations, contrast variation (particularly D2O/H2O) for nuclear structural studies, lack of radiation damage when using crystals of biological molecules such as proteins, and the fidelity to measure nuclear and magnetic diffuse scattering with elastic discrimination.

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In 1932 James Chadwick discovered the neutron using a polonium source and a beryllium target (Chadwick, 1932). In a letter to Niels Bohr dated February 24, 1932, Chadwick wrote: "whatever the radiation from Be may be, it has most remarkable properties." Where it concerns hydrogen-rich biological materials, the "most remarkable" property is the neutron's differential sensitivity for hydrogen and its isotope deuterium. Such differential sensitivity is unique to neutron scattering, which unlike X-ray scattering, arises from nuclear forces. Consequently, the coherent neutron scattering length can experience a dramatic change in magnitude and phase as a result of resonance scattering, imparting sensitivity to both light and heavy atoms, and in favorable cases to their isotopic variants. This article describes recent biomembranes research using a variety of neutron scattering techniques.

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Neutron crystallography is used to locate H atoms in biological materials and can distinguish between negatively scattering hydrogen-substituted and positively scattering deuterium-substituted positions in isomorphous neutron structures. Recently, Hauptman & Langs (2003; Acta Cryst. A59, 250-254) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering H atoms in the structure. Selective-labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of H to D atoms can be precisely controlled. Methyl selective-labeling protocols were applied to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here, the production, crystallization and preliminary neutron analysis of a selectively CH3-protonated deuterated PfRd sample, which provided a high-quality neutron data set that extended to 1.75 A resolution using the new LADI-III instrument at the Institut Laue-Langevin, are reported. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of H atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.

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Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

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Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.

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Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

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The positions of the ordered hydrogen isotopes of a protein and its bound solvent can be determined by using neutron crystallography. Furthermore, by collecting neutron data at cryo temperatures, the dynamic disorder within a protein crystal is reduced, which may lead to improved definition of the nuclear density. It has proved possible to cryo-cool very large Con A protein crystals (>1.5 mm3) suitable for high-resolution neutron and x-ray structure analysis. We can thereby report the neutron crystal structure of the saccharide-free form of Con A and its bound water, including 167 intact D2O molecules and 60 oxygen atoms at 15 K to 2.5-A resolution, along with the 1.65-A x-ray structure of an identical crystal at 100 K. Comparison with the 293-K neutron structure shows that the bound water molecules are better ordered and have lower average B factors than those at room temperature. Overall, twice as many bound waters (as D2O) are identified at 15 K than at 293 K. We note that alteration of bound water orientations occurs between 293 and 15 K; such changes, as illustrated here with this example, could be important more generally in protein crystal structure analysis and ligand design. Methodologically, this successful neutron cryo protein structure refinement opens up categories of neutron protein crystallography, including freeze-trapped structures and cryo to room temperature comparisons.

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The number of protein crystal structures being refined to atomic resolution is increasing each year as well as the size of proteins being studied. There are currently 346 structures in the protein data bank which have been refined to or beyond atomic resolution. The benefits of atomic resolution X-ray data are discussed along with a number of structural examples of biomedically relevant proteins. The complementary role of neutron diffraction will also be discussed.

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Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.

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Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.

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The successful application of the newly developed image-plate neutron Laue diffractometer (LADI) at the Institut Laue-Langevin (ILL), Grenoble, France, for rapid hydrogen-bonding characterization is reported. The case study concerns the promising non-linear optical material zinc (tris)thiourea sulfate (ZTS), which contains 30 atoms in the asymmetric unit and crystallizes in the orthorhombic space group, Pca2(1), a = 11.0616 (9), b = 7.7264 (6), c = 15.558 (1) A [T = 100.0 (1) K]. The results from a 12 h data collection from ZTS on LADI are compared with those obtained over 135 h using the monochromatic four-circle diffractometer D9 at the same reactor source with a crystal 13 times larger in volume. Both studies reveal the extensive hydrogen bonding and other close non-bonded contacts within the material. As expected, the results from D9 are more precise than those obtained from LADI; however, the bond geometry determined from the two experiments is the same within the larger estimated standard deviations. Furthermore, the conclusions drawn from the two studies separately regarding the nature of all supramolecular features are identical. This illustrates that LADI is eminently suitable for rapid characterization of hydrogen-bonded structures by neutron diffraction, with the gain in speed compared with traditional instrumentation being several orders of magnitude.

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Until now, no aspartic proteinase has been subjected to a successful neutron diffraction analysis, owing to the limited size of the crystals. However, the recent development of the neutron Laue technique at ILL and EMBL (Grenoble) has allowed the collection of data to 2.2 A on a complex of endothiapepsin with a transition-state analogue. The objective is to define the positions of the protons at the active site by refinement using the neutron data. In line with work on serine proteinases, where neutron diffraction has provided some of the most definitive data on the catalytic mechanism, it is expected that this work will have a major significance for studies of the aspartic proteinase enzymes.

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The pentameric structure of C-reactive proteins (CRP) has been derived by a combination of automated and manual molecular-replacement techniques. The method is generally applicable to other multimeric assemblies. The highly homologous human serum amyloid P component (hSAP) structure fails to provide a pentameric molecular-replacement solution for CRP. In the absence of a significant signal from an individual protomer, the hSAP structure has been manually modified in terms of protomer assembly to provide the true pentameric model of CRP. The CRP protomers are rotated or twisted by 14 degrees about an axis, through the protomer centre, which is approximately perpendicular to the pentamer radius and the molecular fivefold axis. The results demonstrate clearly that protomers with very similar folds arising from high sequence homology need not necessarily be assembled together in the same way although the symmetry of the resulting oligomer may be maintained. In a curious twist the CRP structure which provided the general CRP model remains unsolved, while the model itself has so far provided the solution of two other CRP structures.

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The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

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Aldehyde reductase, a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of a variety of aldehydes to their corresponding alcohols. The structure of porcine aldehyde reductase-NADPH binary complex has been determined by x-ray diffraction methods and refined to a crystallographic R-factor of 0.20 at 2.4 A resolution. The tertiary structure of aldehyde reductase is similar to that of aldose reductase and consists of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. Unlike aldose reductase, the N epsilon 2 of the imidazole ring of His 113 in aldehyde reductase interacts, through a hydrogen bond, with the amide group of the nicotinamide ring of NADPH.

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Crystals of C-reactive protein from rat have been grown both with and without calcium. Two major components of the protein have been resolved on the basis of their calcium-dependent fine specificity for monophosphate esters. Crystals grown without calcium are tetragonal, space group P42(1)2 with unit cell parameters a = b = 163.81(9)A and c = 125.21(6)A, and diffract X-rays to 3.0 A resolution. The rotation function specifies a molecular 5-fold symmetry axis at 24 degrees away from c in the (110) plane.

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Rotation function studies of two tetragonal crystal forms of human C-reactive protein have confirmed the pentameric structure of the molecule. The two crystal forms have space groups P4122 (I) and P4222 (II) with closely similar unit cells and are often twinned together. Investigation of the crystallization conditions indicates that dissociation heterogeneity has been a major limiting factor in the reproducible growth of good single crystals. The orientation of the pentameric molecule is shown to be almost identical in both forms, about the axial direction omega = 57 degrees, phi = 45 degrees, i.e. 57 degrees away from c in the (110) plane.

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Crystals of C-reactive protein from Limulus polyphemus have been grown both with and without calcium. The space group for the calcium-free crystals is I422 or I4(1)22, and the cell parameters are a = b = 173.33 (4) A, c = 98.81 (3) A. The crystals diffract to at least 2.8 A resolution and are suitable for detailed structural studies.

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Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.

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