Biomedical subjects

E Nogales

Publications and source records attributed to E Nogales.

32 records · Page 2Linked to original sources

PubMed1999

High-resolution model of the microtubule.

A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 A map of the microtubule. The excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the tubulin structure within the microtubule. Long C-terminal helices form the crest on the outside of the protofilament, while long loops define the microtubule lumen. The exchangeable nucleotide in beta-tubulin is exposed at the plus end of the microtubule, while the proposed catalytic residue in alpha-tubulin is exposed at the minus end. Extensive longitudinal interfaces between monomers have polar and hydrophobic components. At the lateral contacts, a nucleotide-sensitive helix interacts with a loop that contributes to the binding site of taxol in beta-tubulin.

Crystallography, X-Ray↗

PubMed1999

Cryo-electron microscopy of GDP-tubulin rings.

Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an "unconstrained" conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozen-hydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules.

Animals↗

PubMed1999

Crystallographic structure of tubulin: implications for dynamics and drug binding.

The structure of tubulin, recently solved by electron crystallography, has given a first look at the molecular basis for some of the properties of tubulin and microtubules that have been observed over the last decades. We discuss how the structure relates to some of these properties, and how inferences about drug binding sites can explain some of the effects of the drugs on tubulin. Microtubules can form a highly dynamic system that requires careful tuning of the stability and properties of tubulin and its interactions with its many ligands. Understanding these interactions can provide fundamental information on the regulation of the microtubule system.

Binding Sites↗

PubMed1998

Structure of the alpha beta tubulin dimer by electron crystallography.

The alphabeta tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is nonexchangeable when it is bound in the alpha subunit, or N site, and exchangeable when bound in the beta subunit, or E site. The alpha- and beta-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of posttranslational modifications. Limited sequence homology has been found with the proteins FtsZ and Misato, which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the alphabeta tubulin dimer fitted to a 3.7-A density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of alpha- and beta-tubulin are basically identical: each monomer is formed by a core of two beta-sheets surrounded by alpha-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.

Amino Acid Sequence↗

PubMed1998

New insights into microtubule structure and function from the atomic model of tubulin.

The structure of tubulin has recently been solved by electron crystallography of zinc-induced tubulin sheets. Because tubulin was studied in a polymerized state, the model contains information on the interactions between monomers that give rise to the alpha beta dimer as well as contacts between adjacent dimers that result in the structure of the protofilament. The model includes the binding site of taxol, an anti-cancer agent that acts by stabilizing microtubules. The present tubulin model gives the first structural framework for understanding microtubule polymerization and its regulation by nucleotides and anti-mitotic drugs at the molecular level.

Amino Acid Sequence↗

PubMed1998

Tubulin and microtubule structure.

Our knowledge of microtubule structure and its relationship to microtubule function continue to grow. Cryo-electron microscopy has given us new images of the microtubule polymerization and depolymerization processes and of the interaction of these polymers with motor proteins. We now know more about the effect of nucleotide state on the structure and dynamic instability of microtubules. The atomic model of tubulin, very recently obtained by electron crystallography, is bringing new insight into the properties of this protein and its self-assembly into microtubules, and promises to inspire new experimental efforts that should lead us to an understanding of the microtubule system at the molecular level.

Image Processing, Computer-Assisted↗

PubMed1998

Tubulin structure: insights into microtubule properties and functions.

The structure of tubulin has recently been determined by electron crystallography, paving the way for a clearer understanding of the unique properties of tubulin that allow its varied functions within the cell. Some of the ongoing work on tubulin can be interpreted in terms of its structure, which can serve to guide future studies.

Colchicine↗

PubMed1998

Tubulin and FtsZ form a distinct family of GTPases.

Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.

Amino Acid Sequence↗

PubMed1997

Visualizing the secondary structure of tubulin: three-dimensional map at 4 A.

We are in the process of determining the structure of tubulin using electron crystallography of zinc-induced, crystalline sheets. We have now extended the resolution to 4 A, and there are many features in the map that appear to show details of the secondary structure. X-ray crystallographers are well aware of the problems of interpreting maps with such limited resolution, and the additional problem of the missing cone of data inherent in electron crystallography may make interpretation even more difficult. To investigate how reliably these maps can be interpreted, we have calculated density maps of a known structure, actin, under conditions similar to those of the tubulin map. Results of these simulations support the limited interpretations we made previously in the 6.5-A maps and the more extensive interpretations we make here in the 4-A map. Most of the secondary structure of the tubulin dimer can now be identified.

Actins↗

PubMed1996

Interpreting a medium-resolution model of tubulin: comparison of zinc-sheet and microtubule structure.

We previously used electron crystallography of zinc-induced two-dimensional crystalline sheets of tubulin to construct a medium-resolution three dimensional (3-D) reconstruction (at 6.5 A) of this protein. Here we present an improved model, and extend the interpretation to correlate it to microtubule structure. Secondary sequence predictions and projection density maps of subtilisin-cleaved tubulin provide information on the location of the C-terminal portion, which has been suggested to be involved in the binding of microtubule-associated proteins. The zinc-sheet tubulin model is compared to microtubules in two ways; comparison of electron diffraction from the zinc-sheets to electron diffraction from microtubules, and by docking the zinc-sheet protofilament 3-D model into a helical reconstruction from ice-embedded microtubules. By correlating the zinc-sheet protofilament to a reconstruction of axonemal protofilaments, we assigned polarity to the protofilament in our model. The polarity assignment together with our model for dimer boundaries and the assignment of alpha- and beta-monomers in our reconstruction, provides a microtubule model where the alpha-monomer crowns the plus- (or fast-growing) end of the microtubule and contact is made in the centrosome with gamma-tubulin via the beta-monomer.

Amino Acid Sequence↗

PubMed1995

The effect of temperature on the structure of vinblastine-induced polymers of purified tubulin: detection of a reversible conformational change.

Addition of the antimitotic drug vinblastine to solutions of purified tubulin induces the formation of helical polymers whose structure and type of aggregation is determined by the concentration of magnesium. While paracrystalline arrangements of single coils are observed at low concentrations of the ion, for concentrations higher than 6 mM free double-coiled spirals are obtained, which are indistinguishable from those obtained in the presence of microtubule-associated proteins (MAPs). This result is consistent with a similar effect of magnesium and MAPs in neutralizing negative charges on the tubulin molecule and so allowing for lateral contacts between protofilaments. The effects that temperature has on the structure of both types of polymers, free spirals or paracrystals, have been monitored using time-resolved X-ray solution scattering. This study shows that a temperature increase: (1) affects the length and lateral aggregation of the spirals in the paracrystalline sample; (2) induces a reversible increase of the helical pitch in both types of polymers that closely follows the temperature change; (3) produces an irreversible aggregation of some of the protein in both types of polymers; and (4) can induce a reversible transformation from one type of structure to the other when the concentration of Mg2+ is in the boundary between the two ranges. We suggest that the changes in pitch are due to a temperature-induced conformational change of the tubulin molecule. This effect may be related to the structural modifications that result in the temperature-induced assembly of microtubules in vitro under normal conditions of assembly.

Animals↗

PubMed1995

Structure of tubulin at 6.5 A and location of the taxol-binding site.

Tubulin, the major component of microtubules, is a heterodimer of two chains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) and with 40-50% identity. The isotypic variety and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work. Structural knowledge of tubulin has been limited to about 20 A from X-ray diffraction of oriented microtubules, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement, and their two-dimensional character is ideal for high-resolution electron microscopy. Here we present a three-dimensional reconstruction of tubulin to 6.5 A resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The alpha- and beta-subunits appear topologically similar, in agreement with their sequence homology. Several features can be defined in terms of secondary structure. An apparent alpha-helical portion, adjacent to both interdimer and inter-protofilament contacts, is tentatively attributed to a segment near the carboxy terminus of the protein. We can assign the alpha- and beta-subunits on the basis of projection studies of the binding of taxol, which show one taxol site per tubulin heterodimer, in agreement with the known stoichiometry of taxol in microtubules. These studies indicate that taxol affects the interaction between protofilaments; to our knowledge, this is the first time that a ligand-binding site has been visualized in the tubulin molecule.

Animals↗

PubMed1992

Low resolution structure of microtubules in solution. Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules.

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.

Alkaloids↗

PubMedDate not supplied

Preservation of 2-D crystals of tubulin for electron crystallography.

Zinc-induced sheets of tubulin are two-dimensional crystalline polymers that constitute an ideal sample for high resolution studies of tubulin by electron crystallography. We show that these 2-dimensional tubulin crystals can be stabilized by taxol against low-temperature depolymerization and degradation with time, easing the way for the preparation of electron microscopy samples. The preservation of the crystals to high resolution has been tested with different embedding media. While glucose-embedded samples diffract poorly, samples embedded in tannin consistently diffract to a resolution of at least 3.5 A. Even better results are obtained by embedding with a combination of tannin and glucose, which improves the flatness of the crystals and allows the collection of isotropic high-resolution data from tilted specimens.

Animals↗