X-ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP), which enzymatically removes a single adenine base from a conserved, surface exposed loop sequence of ribosomal rRNA. We now present unprecedented experimental evidence that PAP can release not only adenine but guanine as well from Escherichia coli rRNA, albeit at a rate 20 times slower than for adenine. We also report X-ray structure analysis and supporting modeling studies for the interactions of PAP with guanine. Our modeling studies indicated that PAP can accommodate a guanine base in the active site pocket without large conformational changes. This prediction was experimentally confirmed, since a guanine base was visible in the active site pocket of the crystal structure of the PAP-guanine complex.     

Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data

The active site of lysostaphin is shown to contain a residue of glutamic acid. As judged by a pK value of 9.2 (with pentaglycine bridges in peptidoglycan of staphylococci as a substrate), another ionogenic residue could be the epsilon-amino group of a lysine. However, the pH value near a negatively charged cell is supposed to be strongly shifted to acidity as compared to the pH of the solution volume. This shifts the enzyme pH dependence curve in solution to alkalinity. Therefore, the other group might be histidine, which is consistent with the X-ray crystallographic data. A similar shift is likely to occur for lysozyme in the case of Micrococcus lysodeikticus cells. Determination of pK of ionogenic groups in the active sites of alkaline enzymes responsible for lysis of negatively charged bacterial cells gives their apparent values because the "pericellular" and "voluminous" values of pH are not coincident.     

A structural analysis of the group II intron active site and implications for the spliceosome

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions

The structure ofE. coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 Å without bound fatty acid and to 2.0 Å and 1.75 Å with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively. The structure ofE. coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 Å with a C16 bacterial fatty acid. Both proteins contain 10 anti-parallel -strands in a+1, +1, +1... motif. The strands are arranged in two -pleated sheets that are orthogonally oriented. In each case, the fatty acid is enclosed by the -sheets and is bound to the proteins by feeble forces. These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity. These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues. Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms. However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

Deprotonation states of the two active site water molecules regulate the binding of protein phosphatase 5 with its substrate: A molecular dynamics study

Protein phosphatase 5 (PP5), mainly localized in human brain, can dephosphorylate tau protein whose high level of phosphorylation is related to Alzheimer's disease. Similar to other protein phosphatases, PP5 has a conserved motif in the catalytic domain that contains two binding sites for manganese (Mn²⁺) ions. Structural data indicate that two active site water molecules, one bridging the two Mn²⁺ ions and the other terminally coordinated with one of the Mn²⁺ ions (Mn1), are involved in catalysis. Recently, a density functional theory study revealed that the two water molecules can be both deprotonated to keep a neutral active site for catalysis. The theoretical study gives us an insight into the catalytic mechanism of PP5, but the knowledge of how the deprotonation states of the two water molecules affect the binding of PP5 with its substrate is still lacking. To approach this problem, molecular dynamics simulations were performed to model the four possible deprotonation states. Through structural, dynamical and energetic analyses, the results demonstrate that the deprotonation states of the two water molecules affect the structure of the active site including the distance between the two Mn²⁺ ions and their coordination, impact the interaction energy of residues R275, R400 and H304 which directly interact with the substrate phosphoserine, and mediate the dynamics of helix αJ which is involved in regulation of the enzyme's activity. Furthermore, the deprotonation state that is preferable for PP5 binding of its substrate has been identified. These findings could provide new design strategy for PP5 inhibitor.     

On the structural significance of the linkage region constituents of N-glycoproteins: an X-ray crystallographic investigation using models and analogs

The linkage region constituents, namely, 2-acetamido-2-deoxy-beta-D-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes. The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents. A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved. This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, psi(N) (O5-C1-N1-C1(')). This also forms the first report on the crystal structure of a model of L-RhabetaAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus. Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a psi(N) value of -114.5 degrees and -121.2 degrees, respectively, deviating maximum from the value of -89.8 degrees reported for the model compound GlcNAcbetaNHAc. The L-Rha and Gal derivatives also show noticeable deviations. The psi(N) values, -89.5 degrees and -91.0 degrees, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcbetaGln and GlcNAcbetaGln, respectively) agree well with those (-93.8 degrees and -89.8 degrees ) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid. However, the rotational freedom about the additional C-C bond would lead to altered rigidity of the linkage region. An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds. The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of beta-1-N-acyl derivatives of GlcNAc. Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

Crystallization and preliminary X-ray crystallographic analysis of the 38-kDa immunodominant antigen of Mycobacterium tuberculosis.

The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.     

Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV–vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.     

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

A novel adenosine triphosphate analog with a heavy atom to target the nucleotide binding site of proteins.

We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.     

Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly

Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 A resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four-stranded parallel beta-sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N-terminal beta-strand interacts with the beta-sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand beta1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the beta-hairpin connecting helices alpha4 and alpha5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface.     

Structure and dynamics of the active site gorge of acetylcholinesterase: synergistic use of molecular dynamics simulation and X-ray crystallography.

The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.     

Kinetic and crystallographic studies on 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of beta-D-glucopyranose, 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K(i) values of 145.2 (+/-11.6), 76 (+/-4.8), and 8.6 (+/-0.7) muM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 A; GPb-benzothiazole, 2.10 A; GPb-benzimidazole, 1.93 A). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (approximately 32 A) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.