MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function.

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.

Cat hemoglobin: pH-dependent cooperativity of oxygen binding.

Cat hemoglobin has a lower cooperativity and oxygen affinity than most mammalian hemoglobins. In contrast to the usual invariance of cooperativity with pH, a rise in cooperativity with pH is predicted by the allosteric model for low-affinity hemoglobins. Such a pH-dependent cooperativity for cat hemoglobin has been found.

Allosteric mechanisms in normal and pathological nicotinic acetylcholine receptors.

Recent chemical and advanced structural studies on site-directed and naturally occurring pathological mutants of individual members of the multigene family of nicotinic acetylcholine receptors have yielded structure-function relationships supporting indirect 'allosteric' interactions between the acetylcholine-binding sites and the ion channel in signal transduction.

An allosteric theory for hemoglobin incorporating asymmetric states to test the putative molecular code for cooperativity.

The two-state (MWC) model for cooperative oxygen binding by tetrameric (alpha2beta2) hemoglobin based on concerted transitions between symmetric states (T and R) is extended to include a third, asymmetric state with one alphabeta dimer possessing high (R-like) oxygen affinity and the other alphabeta possessing low (T-like) oxygen affinity. The asymmetric state is assigned a stability that corresponds to the level reported by Ackers and colleagues in the studies on mixed valence hybrids that led to their proposed "molecular code for cooperativity in hemoglobin." However, this level of stability for the asymmetric intermediates significantly diminishes cooperativity in simulated oxygenation curves, to a degree (Hill n = 2.1) that is no longer compatible with the well-established oxygenation properties of normal ferrous hemoglobin (Hill n approximately 3.0). Therefore, the cyanomet derivatives do not appear to be reliable analogues of intermediate oxygenation states.

Double strand packing in hemoglobin S fibers.

The sickling variant of human hemoglobin, Hb S (beta 6 Glu-->Val), assembles into 14-strand helical fibers composed of seven pairs of double strands. The organization of the helical double strands closely resembles the parallel, half-staggered, linear strand pairs of the crystals of Hb S characterized by Wishner et al. In the crystals, the molecules are arranged such that each possesses a beta 6 Val in contact with a molecule on the opposite strand. In the fibers, the overall hexagonal packing of strands leads to 22 classes of potential contacts between the seven double strands, but the presence of 2-fold helical symmetry reduces these contacts to 11 distinct classes. An analysis of the intermolecular contacts reported by Watowich et al., based on the data of Carragher et al., indicated a loosely packed structure for which only four of the 11 potential classes of contacts between double strands are significant (residues within 5 A). We have recently analyzed the packing based on the results of Dykes et al. and Rodgers et al., and compared the findings with the structure derived from the data of Carragher et al. We find serious differences between the two data sets concerning the packing of double strands. The Dykes-Rodgers data indicate a more closely packed structure in which nine of the 11 potential classes of contacts are within 5 A. Considerations on the stability of certain contacts derived from incomplete fibers, as well as studies of Hb molecules composed of beta S chains and mutant alpha chains, suggest that the structural model with closer packing of the double strands provides a better correlation with the other experimental results.

Contributions of individual molecular species to the Hill coefficient for ligand binding by an oligomeric protein.

New insights into the Hill coefficient (n) as a measure of cooperativity are obtained by resolving Y, the fractional ligand binding to an oligomeric protein, into a series of integral nth-order reactions. For identical sites within a single conformational state, the weighted sum of each reaction multiplied by its net order gives a Hill coefficient at Y = 0.5 of n50 = 1.0, indicative of non-cooperative binding. However, the disappearance of unliganded oligomers (S0) reflects the higher-order reactions, with their weighted sum (for a tetramer) leading to a Hill coefficient at S0 = 0.5 of n50* = -1.27. For an oligomer with two conformational states (such as represented by the T and R states in the Monod-Wyman-Changeux model) capable of generating highly cooperative binding, the same nth-order reactions apply, but with different weights. For oxygen binding to hemoglobin, n50 is resolved into three components with net reaction orders of n = -2, 2, and 4 (with weights of 0.067, 0.15, and 0.754 corresponding, respectively, to the contributions of singly, triply and quadruply liganded molecules) to give n50 = 3.18. However, the cooperativity of the "state" function, R' (the normalized fraction of molecules in the R state), as characterized by n50' (the Hill coefficient at R' = 0.5) is distinct from n50. If the T-R equilibrium lies very far in favor of either state, then even when the two states differ widely in their intrinsic affinity for ligand, the lower limit of cooperativity for Y is n50 = 1.0, but the Hill coefficient for R' cannot fall below n50' = 1.27 (for a tetramer). Hence, the lower limit of n50' is equal to the absolute value of n50* describing the disappearance of S0 for an oligomer with a single conformational state.

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain, glucose or embedding in the presence of tannic acid.

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2(1)2(1)2(1) and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 A. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with non-embedded, negatively stained thin platelet crystals. In addition, good agreement at 20 A resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by low-dose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3 X 7 A as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 A resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.

The innermost chorionic layer of Drosophila. I. The role of chorin octamers in the formation of a family of interdigitating crystalline plates.

The innermost chorionic layer (ICL) within egg shells of Drosophila melanogaster is composed of thin, abutting three-dimensional crystalline plates which form a closed, membrane-like sheath. Collectively, the crystals within the sheath appear to form a family of related three-dimensional crystals in space group C222; however, specimens prepared for electron microscopy are actually two-dimensional crystals in c222. The projected structures of the negatively stained crystals have been studied by minimal dose electron microscopy employing image reconstruction methods. Thin sections indicate that unit cells within the ICL are composed of paired layers; top and bottom layers are related by centrally located 2-fold axes, aligned parallel to the surface of the ICL. The most probable structural unit of the crystals is a tetramer of chorin dimers with a point group symmetry of 222, which is denoted a chorin octamer. Projection maps were computed from average transforms of two-dimensional crystals for delta (the primitive unit cell angle) equal to 84 degrees, 90 degrees and 97 degrees (+/- 1.5 degrees). The maps indicate that the molecular transitions responsible for the observed family of crystals involve concerted intramolecular rearrangements about molecular 2-fold axes. The significance in vivo of the family of crystals within the ICL is not known; however, structural considerations suggest that the observed polymorphism may reflect one facet of an intrinsic bonding flexibility of the ICL octamer that may play a role in the formation of interplate junctions and the assembly of a continuous closed sheath. The ICL may therefore serve as a structural bridge between the vitelline membrane-wax layer and the endochondrial floor, allowing the larva to shed the inner egg shell layers during hatching.

Three-dimensional reconstruction of tubulin in zinc-induced sheets. II. Consequences of removal of microtubule associated proteins.

The three-dimensional structure of zinc-induced tubulin sheets freed of microtubule associated proteins has been determined to 20 A resolution by electron microscopy and image reconstruction. The determination was carried out with porcine brain tubulin separated from microtubule associated proteins by phosphocellulose chromatography. Negatively stained samples were tilted using the goniometer stage of the electron microscope to provide images of the tubulin sheets ranging in tilt from -60 degrees to +60 degrees. The micrographs were digitized and subjected to a cross-correlation analysis to compensate for smooth curvature of the lattice in the sheets. For each angle of tilt, an average unit cell was obtained from the cross-correlation analysis and subsequently a Fourier transform was computed for inclusion in the three-dimensional Fourier data set. The transforms of 47 tilted images plus the average of five untilted sheets were combined and an inverse Fourier transform was applied to give a three-dimensional reconstruction of the microtubule associated protein-free tubulin sheets. Comparison of the protofilament structure in these sheets with the previously published protofilament structure of zinc-induced tubulin sheets containing microtubule associated proteins reveals a number of consequences of the removal of microtubule associated proteins. (1) The extensive internal contact along the protofilament observed in microtubule associated protein-containing tubulin sheets is maintained in microtubule associated protein-free tubulin sheets. (2) In projection, the protofilaments in microtubule associated protein-free tubulin sheets are 2 X 2 A closer together than in microtubule associated protein-tubulin sheets. (3) The deviations of adjacent protofilaments from the plane of the sheets when viewed end-on are more pronounced in the absence of microtubule associated proteins. Differences are also observed at the level of individual tubulin subunits. In particular, the distinct cleft which was found in one class of subunits in tubulin sheets with microtubule associated proteins is absent in the microtubule associated protein-free tubulin sheets. The loss of this cleft and some changes in the shape of the tubulin subunits upon removal of microtubule associated proteins suggest a possible site for the interaction of tubulin with microtubule associated proteins.

Self-association of haemoglobin Olympia (alpha 2 beta 2 20 (B2) Val----Met). A human haemoglobin bearing a substitution at the surface of the molecule.

Oxygenation measurements at equilibrium were carried out for solutions of pure haemoglobin (Hb) Olympia (alpha 2 beta 2 20 (B2) Val----Met) at 200 microM (haem) and revealed a high oxygen affinity (P50 = 4.2 torr at pH 7.20, 25 degrees C) compared to HbA (P50 = 5.6 torr), with the Hill coefficient (eta H) reduced from the normal value of 2.9 to 2.5 for Hb Olympia at neutral pH. 2,3-Diphosphoglycerate and chloride effects were normal, but measurements of the alkaline Bohr effect indicated an excess Bohr effect of about 20% for Hb Olympia. Precise determinations of the oxygen binding curves gave the unexpected finding of a dependence of co-operativity on pH with eta H rising from 2.4 at pH 6.8 to 3.0 at pH 8. Moreover, the Hill coefficient was dependent upon the concentration at alkaline pH and fell to 1.8 in low concentration solutions (approximately 30 microM-haem) of the haemoglobin variant; at this concentration the Bohr effect was normal. This effect of concentration on co-operativity could be accounted for fully by the allosteric model, with introduction of Hb Olympia self-association. In this case the allosteric constant L' for the supramolecular species has the value of the allosteric constant L for the tetramer species, raised to a power equal to the number of molecules in the aggregates and modulated by the ratio of the dissociation constants of the aggregates, DNR/DNT. Model curves with N tetramers per aggregate (where N approximately 2 at pH 7.5 and N approximately 4 at pH 8.0) fully represented the concentration dependence for Hb Olympia of the eta H values and the detailed shape of the experimental curves for eta H as a function of log[y/(1-y)], the first derivative of the Hill plot. These curves are much steeper when supramolecular species are present. Direct measurements of the protein aggregation by centrifugation confirmed the presence of aggregates in the solutions of Hb Olympia. Hb Olympia is therefore one of the few examples of mutant human haemoglobins that self-associate with functional consequences in terms of oxygen binding properties.(ABSTRACT TRUNCATED AT 400 WORDS)

The innermost chorionic layer of Drosophila. II. Three-dimensional structure determination of the 90 degrees crystal form by electron microscopy.

The innermost chorionic layer (ICL) within egg shells of Drosophila is composed of a family of related, thin three-dimensional crystals that form a continuous sheath encapsulating the egg shell lumen. Junctions formed by interdigitating lattices play a central role in the construction of this macroscopic assembly. The three-dimensional structure of a two-dimensional crystal isolated from the ICL, with a primitive lattice angle delta of 90 degrees, has been determined from a complete tilt series of a negatively contrasted specimen at a resolution of 25 A. Inspection of the three-dimensional transform after data merging revealed that the space group is c222 and this symmetry was employed to generate a three-dimensional structure. The basic structural unit of the ICL is an octamer, described formally as a tetramer of dimers with point group symmetry 222. There are two classes of dimer in the octamer designated alpha and beta. The chorin octamer is composed of two classes of bent dimers, which make intramolecular contacts at the top and bottom of the molecule. The alpha-dimers are curved outwards away from the crystallographic 2-fold axis, while the beta-dimers are curved towards the molecular center. In addition, lattice contacts are formed primarily by beta-chorin dimers at both the top and bottom surfaces of the unit cell. The molecular weight of a chorin octamer determined from the analysis is about 6 X 10(5). The conformation of the chorin octamer determined here suggests that permutations of a basic molecular mechanism may be adequate to explain both the observed lattice polymorphisms of the ICL and the formation of interplate junctions necessary for the assembly of the macroscopic sheath.

Analysis of hemoglobin oxygen equilibrium curves. Are unique solutions possible?

As a hemoglobin tetramer is oxygenated, it converts from a form with low ligand affinity to a high-affinity structure. This allosteric transition occurs in partially liganded molecules, typically after two or three ligands are bound. As a result of the co-operative nature of the process, the populations of the partially liganded forms are low. The relative proportions and precise properties of these intermediate substrates are therefore difficult to measure and are subject to controversy. The problem is compounded by compensating effects; for example, over-estimation of the oxygen affinity of triply liganded forms will result in under-estimation of the proportion of these species. Specifically, published values for the oxygen affinity of the triply liganded species vary for identical conditions by a factor of more than 6. In analyses based on the highest affinity values, the triply liganded species virtually disappears. However, this affinity is usually calculated from the last few per cent of the oxygenation curve, and this part of the curve is extremely sensitive to the normalization of the data. We conclude that unique solutions for the ligand affinity and substrate populations may be impossible to achieve, and that unusual mechanisms based on particular combinations of parameters, therefore, should be viewed with caution.

Structural changes in tubulin sheets upon removal of microtubule-associated proteins.

Zinc-induced tubulin sheets without microtubule-associated proteins (MAPs) were assembled from tubulin purified by phosphocellulose chromatography. Large, open sheets were obtained in five-minute incubations at pH 5.7. Electron micrographs of negatively stained sheets showed a protofilament arrangement similar to that observed for zinc-induced sheets with MAPs but with altered lattice parameters. The spacings measured from optical diffraction patterns demonstrated that the protofilaments were 2.2 A closer together in the sheets without MAPs. Each MAP-free sheet was also divided roughly in half by a discontinuity which was parallel to the protofilaments and the relationship between the two domains was deduced from computed transforms. Two-dimensional image processing was carried out by conventional Fourier techniques and by correlation analysis. The correlation analysis improved the reconstructions in this application, with the resolution limited by the inherent properties of the negative stain method to about 14 A. A prominent feature of the computed reconstructions was an alternation of light and dark protofilaments due to differential staining, as revealed by a study of folded sheets. Neighboring protofilaments are related by a 2-fold screw axis, as they are in zinc-induced sheets with MAPs, but the symmetry is masked by the differential staining. The major effect of MAP removal on the structure of the sheets is that the bilobed structure of alternate tubulin subunits is no longer observed. This observation and the closer spacing of protofilaments is consistent with the postulate that some of the MAP molecules lie in the groove between protofilaments and bind to several tubulin dimers.

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression of tubulin in yeast: differences in subunit association.

Microtubules are cytoskeletal organelles composed principally of polymerized alpha beta-tubulin heterodimers. The specific roles and the detailed structures of the individual alpha- and beta-tubulin subunits have not been established, since the conditions necessary for separating the heterodimer result in loss of the subunits' ability to repolymerize. We have overcome this obstacle by constructing plasmids which allow regulated overexpression of individual tubulin subunits in the yeast Saccharomyces cerevisiae under control of the galactose promoter. Overproduction was monitored with alpha- and beta-tubulin-specific antibodies using immunoblotting of cell extracts, and the state of association of the individual subunits in vivo was determined by immunofluorescence microscopy. Cells overproducing only beta-tubulin accumulated fibrous structures associated with the cell membrane, whereas cells overproducing only alpha-tubulin displayed a diffuse signal throughout the cytoplasm. Cells simultaneously overexpressing alpha- and beta-tubulin subunits accumulated membrane-associated, filamentous arrays in which both subunits were incorporated. When cells with the fibrous tubulin-containing structures are treated with zymolyase, a yeast cell wall disrupting enzyme, the fibers appear to splay apart, suggesting that the immunofluorescent rings represent bundles of fibers. Cells overproducing beta-tubulin alone or both alpha- and beta-tubulin were examined at various times after galactose induction, and significant differences were found in the tubulin association state prior to the formation of fibers. For alpha beta-tubulin, fibers form directly from a nuclear structure, whereas beta-tubulin alone first accumulates in the cytoplasm. The differences in patterns of tubulin accumulation and assembly presumably reflect a difference in the intrinsic association properties of the alpha- and beta-subunits.

Paradoxical allosteric effects of competitive inhibitors on neuronal alpha7 nicotinic receptor mutants.

Mutation of the conserved leucine residue, in the second transmembrane domain of the neuronal alpha7 acetylcholine receptor to a threonine (L247T) causes pleiotropic alterations of receptor properties. In this study we examined the effects of competitive inhibitors on the alpha7-L247T physiological responses. While the alpha7 competitive inhibitor dihydro-beta-erythroidine evoked a current comparable to that induced by ACh, other inhibitors such as methyllycaconitine (MLA) and alpha-bungarotoxin (alpha-Bgt) caused a blockade of alpha7-L247T to ACh activation. When applied in the absence of ACh, MLA or alpha-Bgt reduced the cell leakage current, showing that alpha7-L247T displays a significant fraction (10%) of spontaneously open channels. These data can be interpreted in terms of an allosteric model, assuming that the L247T mutant possesses a low isomerization constant L and that MLA and alpha-Bgt stabilize the closed, resting state.

UV laser scanning and fluorescence monitoring of analytical ultracentrifugation with an on-line computer system.

A new optical system for the analytical ultracentrifuge is described, which permits sedimentation to be monitored by fluorescence. The optical system is based on a laser light source, which is focused to a narrow (50 micron) beam. The radical scanning of the beam provides information on the distribution of fluorescing material with distance in the centrifuge cell. Data collection and processing are performed in conjunction with an on-line computer system which sorts incoming fluorescence pulses according to rotor hole and cell sector, averages families of pulses to improve signal to noise ratios and fits the data (in the experiments reported here) to equations to determine sedimentation coefficients. Initial experiments with the system have been performed with bovine serum albumin and indicate that sedimentation can be readily monitored by fluorescence with solutions at concentrations as low as 20 micrograms per ml, with excitation at 257 nm. At these concentrations, the optical density is only in the 0.01 range, too low for experiments with absorption-scanner optical system. Even lower concentrations can be used when fluorescent labels are used with excitation in the visible region of the spectrum. The preliminary studies indicate that fluorescence monitoring of sedimentation will substantially enhance the range of experimental possibilities in ultracentrifugation by improving both the sensitivity of measurements and the discrimination between sedimenting species on the basis of their fluorescence characteristics.

Polarity of the 14-strand fibers of sickle cell hemoglobin determined by cross-correlation methods.

Images of negatively stained fibers of sickle cell hemoglobin have been analyzed by cross-correlation methods. These methods are used to compensate for the curvature and variable repeat distance characteristic of negatively stained fibers. Averaged images obtained by the correlation procedure display considerably more detail than the filtered images obtained earlier by Fourier methods. The averaged images are sufficiently detailed that the back-projection method for obtaining cross-sections can now be applied directly to the correlation-averaged images. This method was used previously to deduce the 14-strand structure on Fourier-filtered images that incorporated only the near-equatorial maxima. In this way the 14-strand structure has been reconfirmed without utilizing the partial Fourier reconstructions employed earlier that might conceivably have introduced spurious features. In addition, application of the correlation procedure with and without inversion of the reference reveals a consistent polarity in all of the fibers examined. The confirmation of the 14-strand structure by a new procedure and the determination of fiber polarity would appear to eliminate the alternative model of the fibers with a 16-strand structure with equal numbers of strands (eight) of each polarity.

Trigonal catalase crystals: a new molecular packing assignment obtained from sections preserved with tannic acid.

The molecular packing of a trigonal crystal form of catalase initially studied by Longley [1] has been re-evaluated. Sections of crystals fixed and preserved with tannic acid were obtained parallel to the (001) and (100) planes. Specimens prepared by either conventional or low temperature embedding maintained 20 A resolution after sectioning. The space group of the crystals is either P3(1)21 or P3(2)21 and the observed unit cell parameters for (001) and (100) are a = b = 174 A, gamma = 119 degrees and b = 189 A, c = 248 A with alpha = 89.5 degrees. Computer-based reconstructions of two principal projections coupled with crystal density measurements allowed the deduction that there is one catalase tetramer per asymmetric unit. The crystal structure consists of 6 molecules packed closely about a common triad screw axis. This interpretation differs from that proposed by Longley [J. Mol. Biol. 30 (1967) 323], because thin sections of embedded crystals were assumed a priori to be positively stained in the early work; in actuality the sections were negatively stained. We also demonstrate that tannic acid fixation can lead to well preserved, positively stained crystal sections under certain conditions.