995 resultados para three-dimensional display
Resumo:
Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.
Resumo:
Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically “blocked” because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.
Resumo:
A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and three-dimensional structure in Asn deamidation is estimated.
Resumo:
p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.
Resumo:
Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.
Resumo:
We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-Å resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.
Resumo:
PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the ∼50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical ∼150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of ∼125–150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.
Resumo:
The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel β helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel β strands, one turn of an α helix, and one turn of a 310 helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.
Resumo:
The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.
Resumo:
Efficient and reliable classification of visual stimuli requires that their representations reside a low-dimensional and, therefore, computationally manageable feature space. We investigated the ability of the human visual system to derive such representations from the sensory input-a highly nontrivial task, given the million or so dimensions of the visual signal at its entry point to the cortex. In a series of experiments, subjects were presented with sets of parametrically defined shapes; the points in the common high-dimensional parameter space corresponding to the individual shapes formed regular planar (two-dimensional) patterns such as a triangle, a square, etc. We then used multidimensional scaling to arrange the shapes in planar configurations, dictated by their experimentally determined perceived similarities. The resulting configurations closely resembled the original arrangements of the stimuli in the parameter space. This achievement of the human visual system was replicated by a computational model derived from a theory of object representation in the brain, according to which similarities between objects, and not the geometry of each object, need to be faithfully represented.
Resumo:
Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.
Resumo:
We present new methods for identifying and analyzing statistically significant residue clusters that occur in three-dimensional (3D) protein structures. Residue clusters of different kinds occur in many contexts. They often feature the active site (e.g., in substrate binding), the interface between polypeptide units of protein complexes, regions of protein-protein and protein-nucleic acid interactions, or regions of metal ion coordination. The methods are illustrated with 3D clusters centering on four themes. (i) Acidic or histidine-acidic clusters associated with metal ions. (ii) Cysteine clusters including coordination of metals such as zinc or iron-sulfur structures, cysteine knots prominent in growth factors, multiple sets of buried disulfide pairings that putatively nucleate the hydrophobic core, or cysteine clusters of mostly exposed disulfide bridges. (iii) Iron-sulfur proteins and charge clusters. (iv) 3D environments of multiple histidine residues. Study of diverse 3D residue clusters offers a new perspective on protein structure and function. The algorithms can aid in rapid identification of distinctive sites, suggest correlations among protein structures, and serve as a tool in the analysis of new structures.
Resumo:
Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.
Resumo:
The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.
Resumo:
The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.