The structure of mouse tumour-necrosis factor at 1.4 A resolution: towards modulation of its selectivity and trimerization.

The 1.4 A resolution structure of recombinant mouse tumour-necrosis factor alpha (mTNF) at 100 K has been determined. The crystals are triclinic, space group P1, with unit-cell parameters a = 48.06, b = 48.18, c = 51.01 A, alpha = 114.8, beta = 103.6, gamma = 91.1 degrees. The structure was refined to a final crystallographic R value of 19.7% (Rfree = 23.3%), including 3477 protein atoms, one 2-propanol molecule, one Tris molecule and 240 water molecules. Throughout the crystal lattice, the trimers are differently packed compared with human TNF, which was crystallized in the tetragonal space group P41212 and refined to 2.6 A resolution. The structures of mTNF and human TNF are very similar, diverging mainly in regions that are either flexible and/or involved in crystal packing. Some loops in mTNF which contain residues important for receptor binding are better resolved than in human TNF, such as the surface-exposed loops 30-34 and 144-147, which are also important for receptor specificity. Compared with human TNFs, the channel formed by the three monomers in mTNF is narrower. One 2-propanol molecule trapped in the trimeric channel could be a lead compound for the design of TNF inhibitors.

The crystal structure of an RNA oligomer incorporating tandem adenosine-inosine mismatches.

The X-ray crystallographic structure of the RNA duplex [r(CGCAIGCG)]2 has been refined to 2.5 A. It shows a symmetric internal loop of two non-Watson-Crick base pairs which form in the middle of the duplex. The tandem A-I/I-A pairs are related by a crystallographic two-fold axis. Both A(anti)-I(anti) mismatches are in a head-to-head conformation forming hydrogen bonds using the Watson-Crick positions. The octamer duplexes stack above one another in the cell forming a pseudo-infinite helix throughout the crystal. A hydrated calcium ion bridges between the 3'-terminal of one molecule and the backbone of another. The tandem A-I mismatches are incorporated with only minor distortion to the backbone. This is in contrast to the large helical perturbations often produced by sheared G-A pairs in RNA oligonucleotides.

Crystallization and preliminary X-ray studies of mouse tumor necrosis factor.

Well diffracting crystals of recombinant mouse tumor necrosis factor (mTNF) have been obtained. The sitting-drop vapor-diffusion method was used to grow crystals suitable for X-ray studies from solutions containing methoxypolyethylene glycol 2000 and isopropanol as precipitants. The crystals belong to the space group P1 with unit-cell dimensions a = 49.40, b = 48.24, c = 51.13 A, alpha = 115.06, beta = 103.32, gamma = 91.27 degrees and one trimer in the asymmetric unit. Crystals are stable to X-rays and diffract beyond 2.0 A. Cooling the crystal during data collection is necessary since crystals dissolve at room temperature.

A curved RNA helix incorporating an internal loop with G.A and A.A non-Watson-Crick base pairing.

The crystal structure of the RNA dodecamer 5'-GGCC(GAAA)GGCC-3' has been determined from x-ray diffraction data to 2.3-A resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson-Crick base pairs make up an internal loop in the middle of the duplex, including sheared G.A pairs and novel asymmetric A.A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34 degrees from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson-Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.

Structure of an RNA double helix including uracil-uracil base pairs in an internal loop.

The crystal structure of the RNA dodecamer 5'-GGACUUUGGUCC-3' has been determined from X-ray diffraction data to 2.6 A resolution. This oligomer forms an asymmetric double helix in the crystal. Four consecutive non-Watson-Crick base-pairs are formed in the middle of the duplex including the first intrahelical U-U (or T-T) pairs observed in an oligonucleotide crystal structure. Two different conformations of U-U pairs are observed in the context of the surrounding sequence. One of these pairs is highly twisted, allowing a bound water to bridge across strands in the major groove. The crystal packing illustrates a new form of RNA helix-helix interaction.

Use of low-molecular-weight polyethylene glycol in the crystallization of RNA oligomers.

We have crystallized a variety of RNA oligonucleotides in a form suitable for X-ray diffraction studies using polyethylene glycol with a low-molecular-weight distribution (PEG 400) as the precipitant. Crystallization experiments on a set of 26 RNA oligomers ranging from eight to 12 nucleotides in length resulted in eight diffraction-quality crystals. Of these eight RNA crystals, six utilized PEG 400 as the precipitating agent. We have also been able to obtain large single crystals of a DNA-RNA hybrid, transfer RNA (two different conditions) and a catalytic RNA from PEG 400 solutions. These results suggest that PEG 400 may be a generally useful alternative to 2-methyl-2,4-pentanediol (MPD) which has, thus far, been the most successful precipitant for DNA oligomers.