Biomedical subjects

Teresa Ruiz

Publications and source records attributed to Teresa Ruiz.

10 recordsLinked to original sources

PubMed2006

Three-dimensional analysis of single particles by electron microscopy: sample preparation and data acquisition.

Electron microscopy of single particles has recently become a very popular field in both biological and material sciences. It might be difficult for a novice researcher new to this field to know how to start tackling a new project. This chapter is designed to serve as a guideline for anyone starting a new project to determine a three-dimensional structure using single-particle techniques. The chapter describes the basic techniques necessary to prepare the samples and acquire the data to calculate a three-dimensional reconstruction in easy-to-understand, step-by-step instructions. It starts with the basic preparation of support films and the usage of a variety of staining techniques needed to assess the quality of the sample and the viability of the project. It ends with a detailed description of vitreous ice preparations designed to acquire high-resolution structural information. Guidelines and tips are given on how to record the best images with an electron microscope. Although this chapter is geared to researchers new to the field, experts might find it not only useful as a reference but also valuable because of the number of practical tips included.

Coloring Agents↗

PubMed2006

Three-dimensional reconstruction of single particles in electron microscopy image processing.

Three-dimensional electron microscopy of single macromolecular assemblies has made large strides forward over the last decade. A large number of image processing techniques have been developed and many have found general distribution. For the proper usage of the wide range of available techniques, a clear concept of all processing steps is essential. This chapter provides step-by-step instruction for the three-dimensional reconstruction of an unknown macromolecule. Where possible, the limitations of the techniques are explained. The chapter attempts to be sufficiently general such so as not to adhere to a single image processing system. Described are alignment techniques for two and three dimensions, classification procedures, and the usage of three-dimensional reconstruction algorithms.

Algorithms↗

PubMed2006

Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen.

Actinobacillus actinomycetemcomitans is a gram-negative, facultative, anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis and adult periodontitis and is the causative agent for other serious systemic infections. Recently, we have identified a protein, EmaA (extracellular matrix protein adhesin A), that mediates the adhesion of A. actinomycetemcomitans to collagen. The conserved sequence and predicted secondary structure suggest that EmaA is an orthologue of the Yersinia enterocolitica adhesin YadA. Electron microscopy examinations of A. actinomycetemcomitans have identified antenna-like protrusions associated with the surface of the bacterium. These structures are absent on emaA mutant strains and can be restored by transformation of the mutant strain with emaA in trans. The loss of these structures is associated with a decrease in the binding of this bacterium to collagen. The antenna-like structures are composed of a long rod that terminates in an ellipsoidal head region. The analysis of these structures using image processing techniques has provided an initial estimate of the overall dimensions, which suggests that the appendages are oligomeric structures formed by either three or four subunits. Together, the data suggest that emaA is required for the expression of novel appendages on the surface of A. actinomycetemcomitans that mediate the adhesion of the bacterium to collagen.

Adhesins, Bacterial↗

PubMed2004

Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1.

A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei Gö1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F. A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3). At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.

Adenosine Triphosphatases↗

PubMed2004

Analysis of heterologous expression of Candida albicans SEC61 gene reveals differences in Sec61p homologues related to species-specific functionality.

The protein secretory pathway has not been studied in depth in Candida albicans despite its essential role in the secretion of enzymes and cell surface components related to the ability of the fungus to colonize the human host. To gain further insight into the elements that participate in the first stages of the secretory process in this fungal pathogen we have isolated and characterized the C. albicans ortholog of SEC61. In other species SEC61 has been shown to encode the core element of the protein translocation apparatus within the ER membrane. The cloned gene appears to be essential for cell viability and encodes a highly conserved protein, very similar to the Sec61p from other yeast species both in sequence and hydropathy profile. However, CaSec61p is not able to complement the thermosensitive-growth phenotype of a Saccharomyces cerevisiae sec61 mutant, even though it is expressed and correctly incorporated into the ER membrane of the transformant cells. We report results indicating that the lack of functional complementation could be related to differences in the primary structure of the cytosolic domain located between the fourth and fifth transmembrane domains of the accepted topological model of Sec61p.

Amino Acid Sequence↗

PubMed2003

Functional implications from an unexpected position of the 49-kDa subunit of NADH:ubiquinone oxidoreductase.

Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.

Amino Acid Sequence↗

PubMed2003

The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites.

Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.

Binding Sites↗

PubMed2002

Resolution of the V1 ATPase from Manduca sexta into subcomplexes and visualization of an ATPase-active A3B3EG complex by electron microscopy.

The effect of the ATPase activity of Manduca sexta V(1) ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V(1) were studied. The V(1) was highly activated in the presence of 0.04-0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A(3)B(3)HEGF and A(3)B(3)EG with a remaining ATPase activity of 52 and 65%, respectively. The hydrolytic-active A(3)B(3)EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V(1) provide an organization of the subunits in the enzyme in the framework of the known three-dimensional reconstruction of the V(1) ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37).

Adenosine Triphosphate↗

PubMed2002

Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.

Fungal Proteins↗

PubMed2002

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.

Base Sequence↗