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Techniques are described for processing neutron distance data for the purpose of deriving information about the three-dimensional organization of macromolecular assemblies.

A map of the 30 S ribosomal subunit is presented giving the positions of 15 of its 21 proteins. The components located in the map are S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S14, S15, S18 and S20.

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6·4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.

Small-angle neutron scattering (SANS) analyses of poly(methyl methacrylate) (PMMA) latexes yielded particle sizes that agree with those determined by a light-scattering technique. Emulsion polymerization of perdeuterated methyl methacrylate (d-MMA) and styrene (d-S) in the presence of preformed PMMA latexes resulted in particles with a core—shell morphology, as determined by SANS analysis for a hollow sphere. The locus of polymerization of the added deuterated monomer is therefore at the particle surface. Shells of d-S were less uniform in thickness than those of d-MMA.

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 x 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H/sub 2/O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, the authors propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H/sub 2/O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H/sub 2/O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H/sub 2/O. The authors propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralizationmore » functions.« less

The large difference in neutron scattering length of hydrogen and deuterium atoms provides a unique tool to study biological macromolecules. These molecules exist in an aqueous environment and have an atomic composition of about 50% hydrogen atoms with the rest being mainly carbon, oxygen and nitrogen. By simply changing the ratio of hydrogen to deuterium the contrast of a sample constituent can be changed without altering the chemical composition. The scattering difference between the hydrogen isotopes has now been used to study structural details of proteins, viruses, nucleic acid protein complexes and membranes. The use of small-angle scattering analysis from proteins in solution is a particularly good example of the power of this new technique. From the first experiments with myoglobin, hemoglobin and TMV solutions in 1968 at the HFBR in Brookhaven it became, however, soon evident that the flux was low and the detection system of a conventional spectrometer was inadequate to measure precisely small differences in the scattering pattern. Since these solution scattering patterns are circularly symmetrical, a development of multidetector systems was the first step to alleviate the need for better data. From an early five detector system [1] the development of linear and two-dimensional position sensitive detectors continues to this day [2–6]. Today, position sensitive area-detectors with an efficiency of 80% and 1.3 mm resolution with an active area of 50 cm × 50 cms are available. These detectors have a counting rate capability of 2 × 10 5 neutrons/s. Other instrumentation developments to facilitate the study of biomolecules include (1) cold moderators, (2) thin film multilayer monochromators [7–9], (3) neutron guides made either of multilayer supermirrors or coated with 58 Ni [10,11], (4) various crystal and mirror focusing devices [12,13]. Some of these features have been incorporated in a small-angle neutron spectrometer at the High Flux Beam Reactor at the Brookhaven National Laboratory. This spectrometer has a unique combination of features such as a high resolution area-detector, adjustable wavelength bandwidth and automated sample changer.

Small angle neutron scattering of both activated and deactivated hexadecameric ribulose bisphosphate carboxylase/oxygenase from spinach revealed that its structure in solution very closely resembles that determined for the deactivated crystalline enzyme from tobacco (Baker, T.S., Eisenberg, D., and Eiserling, F. (1977) Science 196, 293-295). Scattering from both forms of the enzyme in H2O most closely fits that expected from a hollow sphere with an outer radius of 56.4 A and inner radius of 14.3 A. In D2O the enzyme's scattering profiles more closely resembled those of a hollow cylinder with an axial ratio of approximately 1.06 and outer and inner radii of 46.7 and 12.9 A, respectively. Superpositioning of model structures showed that both models determined by neutron scattering correspond quite well to that inferred from x-ray diffraction and electron microscopy of the tobacco enzyme. Sedimentation velocity studies confirmed that no major conformational change occurs upon activation of the spinch carboxylase, both forms exhibiting a sedimentation coefficient, 8(20,w), of 17.8 S. Neutron scattering by activated and deactivated carboxylase from Rhodospirillum rubrum, a dimeric enzyme, best matched that predicted for a solid prolate ellipsoid or cylinder. The estimated dimensions of the model structures correspond strikingly well to those predicted for adjacent pairs of large subunits in the hexadecameric tobacco enzyme.

Experimentally measured scattering data differ from theoretical curves because of departures from point geometry in a real instrument. In a small-angle neutron scattering (SANS) instrument, there are essentially three contributions to the smearing of an ideal curve: (1) the finite divergence of the beam, (2) the finite resolution of the detector, and (3) the poly chromatic nature of the beam. Where the scattering is azimuthally symmetric about the incident beam, indirect Fourier transform (IFT) methods may be used not only to smear an ideal scattering curve, but also to desmear an observed pattern. For experiments where the assumption of azimuthal symmetry cannot be made, alternative procedures based on Monte Carlo (MC) techniques have been developed which simulate the smearing of a given theoretical scattering function. This procedure permits the evaluation of smearing effects in anisotropic systems. Both IFT and MC procedures are illustrated with a range of applications from data taken on the 30 m SANS facility at Oak Ridge National Laboratory. It is shown that for experiments with scattering dimensions < 200 A smearing effects are small (< 5%) and that dimensions up to ~ 1200 A may be accurately resolved after proper evaluation of resolution effects. The procedures described may also be extended to include small-angle X-ray scattering, and an example of one such application is given.

Instrumental smearing effects can result in a significant deviation of measured small-angle scattering data from the ideal cross section. The effect of various contributions to smearing is described. The case of circularly symmetric scattering measured on an instrument collimated by circular apertures is treated in detail. For such measurements, it is shown how known scattering functions can be smeared to allow estimation of parameters by a fit to the observed data, and also how the indirect Fourier transformation method can be used to desmear the observed scattered intensity.

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering measurements in solution. This mapping is the first complete account of the quaternary structure of the small ribosomal subunit to be obtained by any method.

Although the basic facts about the ribosome were already known 40 years ago, elucidating its atomic structure and molecular mechanisms required sheer persistence and the innovative use of new technology and methods. These advances have transformed our understanding of translation in the cell.

The high-resolution structure of a nucleosome core particle gives us our first detailed look at the primary level of eukaryotic DNA organization. The structure reveals the nature of histone–DNA contacts and provides some surprises regarding the histone tails and their possible involvement in higher levels of chromatin organization.

Background: Detailed structural information on ribosomal proteins has increased our understanding of the structure, function and evolution of the ribosome. L14 is one of the most conserved ribosomal proteins and appears to have a central role in the ribonucleoprotein complex. Studies have indicated that L14 occupies a central location between the peptidyl transferase and GTPase regions of the large ribosomal subunit. Results The crystal structure of L14 from Bacillus stearothermophilus has been solved using a combination of isomorphous replacement and multiwavelength anomalous dispersion (MAD) methods. The structure comprises a five-stranded β -barrel, a C-terminal loop region that contains two small α -helices, and a β -ribbon that projects from the β -barrel. An analysis of the structure and the conserved amino acids reveals three surface patches that probably mediate L14–RNA and L14–protein interactions within the ribosome. Conclusion The accepted role of ribosomal proteins is to promote the folding and stabilization of ribosomal RNA. The L14 structure is consistent with this notion, and it suggests that the RNA binds in two sites. One RNA-binding site appears to recognize a distinct region of ribosomal RNA during particle assembly. The second site is smaller and may become occupied during the later compaction of the RNA. The surface hydrophobic patch is a likely site of protein–protein interaction, possibly with L19.

In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms.

Ribosomes are nucleoprotein enzymes which catalyze the formation of polypeptide chains under mRNA control, using aminoacyl tRNAs as substrates—for reviews see Nomura et al. (22) and Chambliss et al. (2). While our knowledge of what these particles do in protein synthesis is satisfactory, our understanding of how they do it is minimal. We still have no idea, for example, what there is about the mechanism of protein synthesis that requires all ribosomes, whatever their source, to be two-subunit enzymes. It is most unlikely that mechanistic questions of even this simple kind will be answered until much more is known about the three-dimensional structure of these particles than is known today.

Ribulosebisphosphate carboxylase/oxygenase requires CO2 and Mg2+ for activation; CO2 reacts with a protein e-amino group to form a carbamate which is stabilized by binding of Mg2+. In the case of the hexadecameric enzyme from spinach, the site of carbamate formation is Lys-201 (Lorimer, 1981). The enzyme from Alcaligenes eutrophus, also hexadecameric, undergoes a substantial change in conformation upon activation as reflected by a decrease in s20,w from 17.5 S to 14.3 S (Bowien, Gottschalk, 1982). To assess the generality of these aspects of activation we have characterized the site of carbamate formation in the carboxylase from Rhodospirillum rubrum, a dimeric enzyme, and have examined the shapes of the activated and deactivated forms of the spinach and R. rubrum enzymes in solution by small angle neutron scattering.

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.

Publisher Summary This chapter describes the low-angle neutron scattering methods to determine the three-dimensional configuration of the proteins of the 30S ribosomal subunit of E. coli . The basic strategy for data collection and analysis has been discussed in this chapter. The chapter also concentrates on improvements in biochemical methods, instrumentation, and data analysis. Scattered intensities and transmissions are measured for the various labeled samples, buffer, empty cell, and blocked-beam background. The raw data for each of these measurements exist as two-dimensional arrays representing the number of counts in each detector element. This chapter presents a schematic diagram of data collection and analysis for measurement of inters protein distances by neutron diffraction. Data inputs include transmission measurements, radially averaged scattered intensifies, and ribosome concentrations. Difference programs calculate interference functions. Sine-Fourier inversion of interference functions to obtain P(r) distribution functions follows. Finally, a large set of the P(r) second moments is used as input to mapping programs to obtain centroid coordinates and the radius of gyration of each small subunit protein.

A small angle x-ray scattering study of a rhodopsin-lipid complex in hexane demonstrates the presence of two distinct particle populations with corresponding radii of gyration of approximately 22 and 160 A. A model based on the existence of inverted lipid micelles surrounding the protein polar moieties while the protein hydrophobic domains act as cross-links between the inverted micelles is presented. It accounts for the results herewith reported and explains most of the properties hitherto characterized for the rhodopsin-lipid complex in hexane.

The methods involved in determining the 850 kDa structure of the 30S ribosomal subunit from Thermus thermophilus were in many ways identical to those that are generally used in standard protein crystallography. This paper reviews and analyses the methods that can be used in phasing such large structures and shows that the anomalous signal collected from heavy-atom compounds bound to the RNA is both necessary and sufficient for ab initio structure determination at high resolution. In addition, measures to counter problems with non-isomorphism and radiation decay are described.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA-binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X-ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in 'tramline' complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild-type recombinant GH5. They still bind to H1/H5-depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.

Native human plasminogen has a radius of gyration of 39 angstroms. Upon occupation of a weak lysine binding site, the radius of gyration increases to 56 angstroms, an extremely large ligand-induced conformational change. There are no intermediate conformational states between the closed and open form. The conformational chang is not accompanied by a change in secondary structure, hence the closed conformation is formed by interaction between domains that is abolished upon conversion to the open form. This reversible change in conformation, in which the shape of the protein changes from that best described by a prolate ellipsoid to a flexible structure best described by a Debye random coil, is physiologically relevant because a weak lysine binding site regulates the activation of plasminogen.