Analysis of models of irregular shape by solution X-ray scattering: the case of the 50S ribosomal subunit from E. coli

Structural models of biological macromolecules can be tested by comparing calculated and experimental solution scattering curves. We have developed an approach for computing scattering shape functions at medium resolution from models proposed on the basis of other techniques such as electron microscopy. We present the results obtained with the 50S ribosomal subunit from Escherichia coli; two models are considered, one proposed by Lake (1976), the other one by Tischendorf et al. (1975). Although the two models are similar in many respects, their scattering shape functions are significantly different. The comparison with the experimental scattering curve allows us to check the scale of the models and, after scaling, to quantitate the agreement between the observed and the calculated curves. Finally, it can provide a starting point for the structural interpretation of the X-ray data.     

Serine acetyltransferase from Escherichia coli is a dimer of trimers

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.     

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.     

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit

A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E. coli is proposed. The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering. The model is consistent with nearly all of the published experimental findings on the structure of domain III.     

Physical characteristics of ribosomal protein S4 from Escherichia coli

A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric. A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model. This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies. This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality. Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.     

Structure of a beheaded 30 S ribosomal subunit from Thermus thermophilus.

The 22 S ribonucleoproten particles containing the 5' (body) and the central (platform) domains of the Thermus thermophilus 30 S subunit has been studied by sedimentation, neutron scattering and electron microscopy. The RNP particles have been obtained by oligonucleotide-directed cleavage of 16 S RNA with ribonulease H in the region of the 900th nucleotide of the protein-deficient derivatives of the 30 S subunits. It is shown that these RNP particles are very compact, though their form and dimensions differ slightly from those expected from the electron microscopy model of the 30 S subunit beheaded by computer simulation. The particles are subdivided into two structural domains whose mutual arrangement differs from that of the corresponding morphological parts of the native 30 S subunit. Electron microscopy demonstrates that the mutual arrangement of domains in the RNP particles is not strictly fixed suggesting that interaction with the third domain of the 30 S subunit is a requisite for their correct fitting.     

The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA.

We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA. To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure. The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA. The root mean square distance between cross-link sites and protein centres is 32 A. The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins. The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.     

Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer

We have used F?rster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 S subunits and 70 S ribosomes from X-ray and cryo-electron microscope structures, and the platform is predicted to undergo a clock-wise rotation upon association.     

Reinvestigation of the shape and state of hydration of the skeletal myosin subfragment 1 monomer in solution

Hydrodynamic calculations lead to the conclusion that chymotryptic (or ethylenediaminetetraacetic acid) myosin S1 in solution (hydrated), at 1-5 degrees C, can be modeled as a prolate ellipsoid, with an axial ratio lying between p = 1.0 and 2.5 (major axis between 100.5 A, for p = 1.0, and 162.5 A, for p = 2.5). The degree of hydration is considerable (1.24 g/g for p = 2.5 and 2.02 g/g for p = 1.0). The dehydrated myosin head is pear-shaped under the electron microscope, and its narrowest part is located near the junction with the tail [Elliott, A., & Offer, G. (1978) J. Mol. Biol. 123, 505-519]. Mendelson & Kretzschmar [Mendelson, R. A., & Kretzschmar, K.M. (1980) Biochemistry 19, 4103-4108] have shown that the pear-shaped molecule does not predict the experimental X-ray scattering curve. Nor is this model able to predict the hydrodynamic values. The three-dimensional model for S1 used by Mendelson and Kretzschmar gives a rather good fit to the experimental X-ray scattering curve, but it does not predict the hydrodynamic values. In order to try to reconcile the three models and to fit the X-ray scattering curve and the hydrodynamic data, we suggest that, in solution, the S1 monomer has the shape of a prolate ellipsoid and that an inclusion of bound water exists at one extremity of the protein. The rest of bound water surrounds the protein. As first approximation, the dry protein and the hole are assumed to have the same shape as the hydrated molecule (prolate ellipsoid; p).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the Top a-t component of alfalfa mosaic virus. A non-icosahedral virion

Neutron-scattering in combination with quasi-elastic light-scattering and electron microscopy was used to derive a model for the capsid structure of the Top a-t component of alfalfa mosaic virus (AMV-Ta-t). In the electron microscope, AMV-Ta-t appears as an irregular ellipsoidal particle with apparent dimensions 275 (+/- 31) A X 225 (+/- 22) A. Assuming that the particles are monodisperse, model calculations show that the neutron-scattering data are best explained by an oblate ellipsoidal shape for the virion with external dimensions 284 A X 284 A X 216 A. Based on this result, and in combination with the known composition of the virion, it is suggested that the capsid structure could be based on a deltahedron with 52 pointgroup symmetry and comprising 120 subunits. Such a model would imply a greater deviation from equivalent subunit interactions than normally necessary in icosahedral capsids. The neutron and photon correlation data, however, do not allow us to rule out the possibility that Top a-t is a slightly polydisperse preparation of irregular prolate shapes with mean dimensions 312 A X 232 A X 232 A. Both possibilities support the concept of alfalfa mosaic virus coat protein being capable of a wide range of intersubunit interactions, this flexibility resulting in considerable polymorphism in capsid structures.     

Haloarcula marismortui 50S subunit-complementarity of electron microscopy and X-Ray crystallographic information

The large 50S subunit of the Haloarcula marismortui 70S ribosome was solved to 19 A using cryo-electron microscopy and single particle reconstruction techniques and to 9 A using X-ray crystallography. In the latter case, phases were determined by multiple isomorphous replacement and anomalous scattering from three heavy atom derivatives. The availability of X-ray and electron microscopy (EM) data has made it possible to compare the results of the two experimental methods. In the flexible regions of the 50S subunit, small differences in the mass distribution were detected. These differences can be attributed to the influence of packing in the crystal cell. The rotationally averaged power spectra of X-ray and EM were compared in an overlapping spatial frequency range from 60 to 13 A. The resulting ratio of X-ray to EM power ranges from 1 to 15, reflecting a progressively larger underestimation of the Fourier amplitudes by the electron microscope.     

Structure of myosin subfragment 1 from low-angle X-ray scattering

The X-ray scattering pattern produced by a solution of myosin subfragment 1 has been measured to a resolution (Bragg spacing) of 2 nm. We find that for subfragment 1 (S1) prepared by limited papain digestion in the presence of ethylenediaminetetraacetate the radius of gyration is 3.28 +/- 0.06 nm, the volume is 151 +/- 6 nm3, the surface area is 330 +/- 15 nm2, and the length of the maximum chord is 12.0 +/- 1.0 nm. The theoretical scattering patterns from several objects of uniform electron density have been calculated and compared with the observed scattering produced by S1. The recent three-dimensional electron micrograph reconstruction of S1-decorated actin by J. Seymour and E. O'Brien (private communication) generated the calculated pattern that best fit the observed scattering. This fit strongly suggests that this reconstruction resembles subfragment 1. The good correspondence between an S1 structure derived when S1 is attached to actin and a study of free S1 in solution strongly suggests that binding to actin does not grossly distort the shape of S1. This is consistent with the notion that S1 changes its orientation on actin, rather than its shape, in order to generate the contractile force in muscle.     

我国睡莲科花粉形态的研究

本文对我国睡莲科5属11种2变种的花粉形态进行了系统的研究。每种花粉都通过光学显微镜和扫描电镜进行观察和照相。同时对莲和白睡莲二种花粉作了超薄切片,对它们的外型结构进行了透射电镜的观察和照相。并结合有关分类学和孢粉学资料,讨论了本科的原始性、异质性及演化上的问题。     

Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.

A method is proposed to restore ab initio low resolution shape and internal structure of chaotically oriented particles (e.g., biological macromolecules in solution) from isotropic scattering. A multiphase model of a particle built from densely packed dummy atoms is characterized by a configuration vector assigning the atom to a specific phase or to the solvent. Simulated annealing is employed to find a configuration that fits the data while minimizing the interfacial area. Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.     

Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.     

Small-angle X-ray study on the quaternary structure of erythrocruorin from Helisoma trivolvis

The erythrocruorin from the aquatic snail Helisoma trivolvis was studied in sodium phosphate buffer at pH 6.7 by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 9.4 ± 0.1 nm and maximum dimension 29 ± 1 nm. A model which fits the experimental data well is presented. The overall shape is best described by a slightly ellipsoidal shape with a hole in the centre. A model consisting of 12 subunits forming a slightly ellipsoidal shape fits very well all scattering data.