John T. Finch

Structure of yeast phenylalanine tRNA at 3 A resolution.

The structure of a tRNA has been determined by isomorphous replacement. Some of the interactions which maintain the tertiary structure are of a novel type. Our model differs significantly from one which has recently been proposed.

The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements.

The nuclear hormone receptors are a superfamily of ligand-activated DNA-binding transcription factors. We have determined the crystal structure (at 2.4 A) of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA. The protein binds as a symmetrical dimer to its palindromic binding site consisting of two 6 bp consensus half sites with three intervening base pairs. This structure reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs. Since all nuclear hormone receptors recognize one or the other of these two consensus half site sequences, this recognition mechanism applies generally to the whole receptor family.

Structure of nucleosome core particles of chromatin.

Crystals have been obtained of nucleosome cores and analysed by X-ray diffraction and electron microscopy. The core is a flat particle of dimensions about 110 × 110 × 57 Å, somewhat wedge shaped, and strongly divided into two ‘layers’, consistent with the DNA being wound into about 1¾ turns of a fiat superhelix of a pitch about 28 Å. The organisation of the DNA can be correlated with the results of enzyme digestion studies. A change in the screw of the DNA double helix on nucleosome formation can be deduced.

Amyloid fibers are water-filled nanotubes

A study of papers on amyloid fibers suggested to us that cylindrical β-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-l-glutamine fits a cylindrical sheet of 31 Å diameter made of β-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-Å-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 Å thick. They have a measured mass of 1,450 Da/Å, compared with 1,426 Da/Å for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 Å. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical β-sheets. Fibers of Alzheimer Aβ fragments and variants are probably made of either two or three concentric cylindrical β-sheets. Our structure of poly-l-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37–40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The Aβ peptide of Alzheimers disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.

Image reconstructions of helical assemblies of the HIV-1 CA protein

The type 1 human immunodeficiency virus (HIV-1) contains a conical capsid comprising ∼1,500 CA protein subunits, which organizes the viral RNA genome for uncoating and replication in a new host cell. In vitro, CA spontaneously assembles into helical tubes and cones that resemble authentic viral capsids. Here we describe electron cryo-microscopy and image reconstructions of CA tubes from six different helical families. In spite of their polymorphism, all tubes are composed of hexameric rings of CA arranged with approximate local p6 lattice symmetry. Crystal structures of the two CA domains were ‘docked’ into the reconstructed density, which showed that the amino-terminal domains form the hexameric rings and the carboxy-terminal dimerization domains connect each ring to six neighbours. We propose a molecular model for the HIV-1 capsid that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal ‘defects’.

Crystal structure of uncleaved ovalbumin at 1.95 A resolution.

Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.

Familial dementia caused by polymerization of mutant neuroserpin

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutation, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Sixteen groups of plant viruses.

Abstract Sixteen groups of plant viruses are described. The type members and main characteristics of each group are listed. The extension of this system to other plant viruses is discussed, and it is recommended that only well studied viruses should be grouped. Names are proposed for twelve of the groups: bromovirus, carlavirus, caulimovirus, comovirus, cucumovirus, nepovirus, potexvirus, potyvirus, tobamovirus, tobravirus, tombusvirus, and tymovirus. We suggest that the value of these names should be tested by using them for an experimental period.

Structure of viruses of the papilloma-polyoma type: I. Human wart virus

It has been reported by M. G. Williams, Howatson & Almeida (1961) that the outer shell (“capsid”) of human wart virus consists of 42 morphological units (“capsomeres”) arranged with icosahedral symmetry. This model follows a similar proposal for the structure of polyoma virus by Wildy, Stoker, Macpherson & Horne (1960) . A critical discussion is given of this work and of that of Howatson & Crawford (1963) on disrupted particles, and it is shown that there are no grounds for regarding the 42-structure as established. The experimental work reported here and in later papers of this series demonstrates that the model is incorrect for the papilloma-polyoma family of viruses. In this paper we describe the results of an examination of human wart virus in the electron microscope, using the method of negative staining. Our investigation has inevitably been bound up with an interpretation of which surfaces or sides of the particle are being revealed by the negative staining. We have found a proportion of predominantly “one-side” images and also some clear examples of “two-side” images, both of which can be explained on the basis of the same model. We have analysed the arrangement of regularly disposed morphological units that can be discerned on the surface of dominantly “one-side” particle images in terms of the theory of Caspar & Klug (1962) , which provides a complete classification for icosahedral protein shells. The outer shell is found to have the symmetry of the T = 7 ( dextro ) icosahedral surface lattice and is composed of 72 morphological units. A shell of this type is built of 420 (= 7 × 60) ultimate structure units. This class of icosahedral surface lattice has not hitherto been recognized for any other virus, but its possible existence was predicted by the theory. The structure belongs to a skew class and can therefore exist in either right-handed or left-handed form. The images of all the particles the structures of which we have been able to analyse appear to have the same hand. This fact gives a strong indication that one side of these particles is being contrasted more strongly than the other. It was necessary to discover which side this was in order to establish the absolute hand of the structure. By means of stereomicrography we have obtained strong indications that, under our conditions, the predominant contrast arises from the negative stain lying between the particle and the carbon substrate. It has thus been concluded that the hand is dextro , and we have used a convention of printing which shows the particle as it would be seen from the outside. The proportion of particle images that can be analysed as above is rather small. The surface detail shown up in the majority of particle images is too confused to reveal the structure. We believe that this confusion arises largely not from true disorder within these virus particles but because, in the electron microscope images, both sides of the particles are being contrasted to a varying extent. We have been able to interpret some of the more distinctive appearances in terms of such a superposition of detail from the two sides of a T = 7 surface lattice. A particularly striking appearance is that of a particle, roughly triangular in profile, with three normal morphological units surrounded by terraces of smaller apparent units. We have analysed this appearance as arising from the superposition of approximately equally contrasted detail from both sides of a particle which has the T = 7 surface structure and is viewed down a 3-fold axis. This analysis also yields rough co-ordinates for the 420 structure units making up the outer shell. The units are situated at a distance from the local 6-fold axes of approximately one-third the edge length of the basic triangular cell of the surface lattice, but the angular co-ordinates remain somewhat uncertain. The measured distance between adjacent morphological units in the central region of one-sided images (i.e. those units viewed approximately end-on) is 97 ± 6 A, a lattice distance which, for T = 7, would correspond to a spherical surface of diameter 435 ± 25 A. Since the maximum diameter is 560 A, this implies that the level at which the substructure is revealed and the effective or mean contrast is developed lies at a distance of about 60 A below the outermost points of the particle. The diameter of this “apparent surface of end-on contrast” must be distinguished from the diameter obtained by measuring the distance between the edges of the particle image. For human wart virus, the latter is ∼ 530 A.

Regulation of skeletal muscle contraction: II. Structural studies of the interaction of the tropomyosin-troponin complex with actin☆☆☆

Abstract The structure of the actin-tropomyosin-troponin complex was studied by optical diffraction and three-dimensional reconstruction from electron micrographs. Optical diffraction experiments suggest that troponin is attached every 385 A along the actin-tropomyosin helix, which has a pitch of about 2 × 350 A. Three-dimensional reconstruction studies suggest that the tropomyosin strands lie off the centre line in each of the two long-pitch grooves of the actin 2-start helix, in such a way that each tropomyosin molecule apparently contacts only one of the two actin helical chains which define those grooves. Thus, these two actin chains might be separately controlled by interaction with distinct tropomyosin moieties. Under appropriate conditions (in the absence of Ca2+ and in the presence of pyrophosphate), the structure of the complex of subfragments of myosin with actin was significantly altered by the presence of tropomyosin-troponin.