SEARCH · PubMed Health

Results for “Cryoelectron Microscopy”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources

PubMed2000

Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy.

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions. Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.

Apolipoprotein B-100↗

PubMed1999

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Animals↗

PubMed2001

Single-particle study of protein assembly.

A study of protein assembly in solution with single-particle imaging and reconstruction techniques using cryoelectron microscopy is reported. The human glutamine synthetase enzyme, important in brain metabolism, and previously assumed to be assembled into a homogeneous quaternary structure, is found to be heterogeneous, with three oligomeric states that co-exist at room temperature. This result corrects an old structural and kinetic model determined by ensemble averaging techniques that assumed a homogeneous system. Unexpectedly fast protein dissociation kinetics results from a stabilized transition state.

Brain↗

PubMed2000

A functional water channel protein in the pathogenic bacterium Brucella abortus.

The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium Brucella abortus. The gene was mapped on the large chromosome of B. abortus. It is flanked by one upstream and two downstream copies of the Brucella repeated sequence Bru-RS. Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport. Expression in Xenopus oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel. Glycerol uptake experiments in E. coli also showed that the protein is not able to transport glycerol.

Amino Acid Sequence↗

PubMed2001

RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells.

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.

Amino Acid Motifs↗

PubMed2006

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition.

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.

Algorithms↗

PubMed2003

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data.

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 x 6-mer) in the 75kDa range. A 3D reconstruction of the 1 x 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as alpha-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P.elephas and the American spiny lobster Panulirus interruptus. Comparison of the P.elephas hemocyanin electron microscopy (EM) density map with the known P.interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597-605) and the C-terminal end (654-657), which are absent in the available P.interruptus X-ray data.

Amino Acid Sequence↗

PubMed1999

Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function.

alphaB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in alphaB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using alpha-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G alphaB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G alphaB-crystallin to unfolding alpha-lactalbumin enhanced the kinetics and extent of its aggregation. R120G alphaB-crystallin became entangled with unfolding alpha-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G alphaB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G alphaB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G alphaB-crystallin has decreased beta-sheet secondary structure and an altered aromatic residue environment compared with wild-type alphaB-crystallin. The apparent molecular mass of R120G alphaB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type alphaB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G alphaB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of alphaB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Alcohol Dehydrogenase↗

PubMed2003

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle.

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of approximately 12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change has been confirmed as has the rod to sphere micronemal shape change. Intact micronemes released directly from sporozoites exclude negative stain and appear as smooth-surfaced electron transparent particles. Biochemically purified rod-shaped C. parvum micronemes are shown to be fragile organelles that inevitably undergo variable damage during isolation, storage and subsequent specimen preparation for EM study. In the absence of glutaraldehyde fixation, damaged micronemes allow the negative stain to enter and loose their contents and during storage undergo a rod-to-sphere shape transformation. Glutaraldehyde-fixed micronemes maintain the rod shape; intact fixed micronemes still exclude negative stain but damaged micronemes reveal a complex quasi-helical arrangement of internal protein within the rod-like micronemes. Loss of this internal organized structure appears to be responsible for the micronemal shape change. This interpretation has been advanced from mutually supportive data obtained from cryoelectron microscopy of unstained vitrified samples, conventional air-dry negative staining and cryo-negative staining. Attempts to biochemically solubilize the micronemal content by lysis and ultrasonication, and separate it from the micronemal membranes, have so far met with limited success as the internal material tends to remain as a disorganized cluster of particles upon release.

Animals↗

PubMed2003

Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses.

The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

Amino Acid Sequence↗

PubMed2004

The role of molecular conformation on tumor uptake of polymeric contrast agents.

RATIONALE AND OBJECTIVE: We wanted to test the hypothesis that linear extended polymers are able to translocate across compromised endothelia to a much higher degree than polymers that are in a coiled configuration. The basis for this hypothesis is the concept of polymer reptation, a process allowing linear polymers to move around fixed obstacles and through small pores. MATERIALS AND METHODS: The conjugation of polylysine with DTPA was varied from 55% to 95% of lysine moieties for a fixed polymer degree of polymerization. The variable conjugation constructs were assessed in their ability to enhance tumor signals in an animal model. The blood circulation time was also assessed, as was the proton relaxivity of the constructs. RESULTS: The relaxivity values of the variable conjugation constructs indicated that the conformation of the highly conjugated polymers was consistent with an extended conformation. Images of the highly conjugated constructs with cryoelectron microscopy showed structures also consistent with an extended peptide chain conformation. These constructs gave tumor signal enhancements 10 times larger than observed for constructs of lower conjugation at the same injection dose. CONCLUSIONS: The conformation of polymer agents is of decisive importance in the imaging efficacy of tumors. Polylysine conjugates of Gd-DTPA, at conjugation levels of 90% or higher, assume an extended linear conformation, which enables them to translocate across tumor endothelium more effectively than less highly conjugated polymers.

Adenocarcinoma↗

PubMed1998

Co-reconstitution and co-crystallization of phospholamban and Ca(2+)-ATPase.

Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail.

Animals↗

PubMed1999

Review: rhinoviruses and their ICAM receptors.

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen or macrophage-1 antigen. However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses.

Animals↗

PubMed2001

DNA in human and stallion spermatozoa forms local hexagonal packing with twist and many defects.

In human and other mammal sperm nuclei, DNA is packed in a highly condensed state, the structure of which remains unsolved. Cryoelectron microscopy of vitrified sections provides a first direct view of the local arrangement of the nucleoprotamine filament. DNA aligns in parallel in layers and its orientation rotates along a single-twist direction as in a cholesteric liquid crystal. The structure contains numerous defects, which introduce locally double-twist configurations. Destruction of the SS bonds with dithiotrehitol relaxes the twist and favors the extension of the hexagonal close packing of the filaments, though keeping constant their interfilament distance.

Animals↗

PubMed2006

Are liquid crystalline properties of nucleosomes involved in chromosome structure and dynamics?

Nucleosome core particles correspond to the structural units of eukaryotic chromatin. They are charged colloids, 101 Angstrom in diameter and 55 Angstrom in length, formed by the coiling of a 146/147 bp DNA fragment (50 nm) around the histone protein octamer. Solutions of these particles can be concentrated, under osmotic pressure, up to the concentrations found in the nuclei of living cells. In the presence of monovalent cations (Na(+)), nucleosomes self-assemble into crystalline or liquid crystalline phases. A lamello-columnar phase is observed at 'low salt' concentrations, while a two-dimensional hexagonal phase and a three-dimensional quasi-hexagonal phase form at 'high salt' concentrations. We followed the formation of these phases from the dilute isotropic solutions to the ordered phases by combining cryoelectron microscopy and X-ray diffraction analyses. The phase diagram is presented as a function of the monovalent salt concentration and applied osmotic pressure. An alternative method to condense nucleosomes is to induce their aggregation upon addition of divalent or multivalent cations (Mg(2+), spermidine(3+) and spermine(4+)). Ordered phases are also found in the aggregates. We also discuss whether these condensed phases of nucleosomes may be relevant from a biological point of view.

Animals↗

PubMed2003

Flexibility of the adenovirus fiber is required for efficient receptor interaction.

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

Adenoviridae↗

PubMed2026

Native yeast kinetochore structures identify an essential inner kinetochore interaction.

Kinetochores must accurately assemble on centromeres for faithful chromosome segregation. Although a conserved centromeric nucleosome is essential for kinetochore assembly, budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, perhaps due to its intrinsic rigidity. To better understand yeast inner kinetochore assembly, we develop a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We perform cryoelectron microscopy on the purifications and generate density maps of four separate inner kinetochore complexes, two of which have not been previously visualized and may represent intermediate assemblage states. We identify an Ndc10 trimerization domain that engages centromeric DNA and a pair of CBF3 complexes and is associated with substantial bending of centromeric DNA. Ndc10 trimerization is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates centromeric DNA bending to stabilize the centromeric nucleosome and inner kinetochore.

Kinetochores↗

PubMed2000

A major conformational change in p97 AAA ATPase upon ATP binding.

AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.

Adenosine Diphosphate↗