[Show abstract][Hide abstract] ABSTRACT: A widely studied achiral porphyrin, which is highly soluble in aqueous solutions (TPPS4 ), is shown to self-assemble into helical nanotubes. These were imaged by electron cryo-microscopy and a state-of-the-art image analysis allows building a map at ~5 Å resolution, one of the highest obtained so far for molecular materials. The authors were able to trace the apparent symmetry breaking to existing nuclei in the "as received samples", while carefully purified samples show that both handnesses occur in equal amounts.
[Show abstract][Hide abstract] ABSTRACT: Interpretation of the structural information in cryomicroscopy images recorded on film or CCD camera requires a precise knowledge of the electron microscope parameters that affect image features such as magnification and defocus. Magnification must be determined in order to combine data from different images in a three-dimensional reconstruction and to accurately scale reconstructions for fitting with atomic resolution models. A method is described for estimating the absolute magnification of an electron micrograph of a frozen-hydrated specimen using horse spleen apoferritin as a standard. Apoferritin is a widely available protein complex of known structure that may be included with the specimen of interest and imaged under conditions identical to those used for imaging other biological specimens by cryomicroscopy. The sum of the structure factor intensities of images of randomly-oriented apoferritin particles shows three low resolution peaks to 25Å that arise from the hollow ball structure of apoferritin. Comparison of peak positions of the experimental intensities with structure factor intensities of an atomic model of apoferritin determined by X-ray crystallography provides a scale factor for estimating the absolute magnification of the micrograph. We compare the magnification estimate using apoferritin to that obtained with tobacco mosaic virus, another common magnification standard for cryomicroscopy. We verify the precision of the method by acquiring images with a systematic variation of magnification.
[Show abstract][Hide abstract] ABSTRACT: The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T=3 and T=4 icosahedral shells, and atomic coordinates are available for the T=4 shell. Since the modified protein assembles predominantly into T=3 shells, a quasi-atomic model of the native T=3 shell was made. The spikes in this T=3 structure resemble those in T=4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.
[Show abstract][Hide abstract] ABSTRACT: We describe a multi-hole condenser aperture for the production of several electron beams in the transmission electron microscope (TEM) making it possible to simultaneously image and irradiate spatially separated regions of a specimen. When the specimen is a thin film of vitreous ice suspended over a holey carbon film, simultaneous irradiation of the adjacent carbon support with the off-axis beam compensates for some of the effects of charging in the image formed by a beam irradiating only the ice. Because the intervening region is not irradiated, charge-neutralization of frozen-hydrated specimens can occur by a through-space mechanism such as the emission of secondary electrons from a grounded carbon support film. We use paraxial charge compensation (PCC) to control the amount of charge build-up on the specimen and observe the effects of charge on images. The multi-hole aperture thus provides a tool for investigating the mechanism of charging and charge mitigation during the imaging of radiation sensitive biological specimens by cryomicroscopy.
[Show abstract][Hide abstract] ABSTRACT: Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.
Proceedings of the National Academy of Sciences 06/2010; 107(23):10685-90. DOI:10.1073/pnas.1002123107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.
Proceedings of the National Academy of Sciences 09/2009; 106(41):17407-12. DOI:10.1073/pnas.0902977106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacteriophage phi12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae.
We present structural studies of cystovirus phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7.
In conclusion, we have determined that the phi12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis.
PLoS ONE 02/2009; 4(9):e6850. DOI:10.1371/journal.pone.0006850 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol gammaA, and of the holoenzyme including a dimeric accessory factor, pol gammaB(2), permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L(549) of pol gammaA upon binding of the accessory subunit. This interaction region is near residue 467 of pol gammaA, where a disease-related mutation has been reported to impair binding of the B subunit. One pol gammaB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol gamma is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.
The EMBO Journal 11/2007; 26(19):4283-91. DOI:10.1038/sj.emboj.7601843 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FokI is a type IIS restriction endonuclease that recognizes the 5'-GGATG-3' sequence and cleaves non-specifically at 9 and 13 base-pairs away on the top and bottom strands, respectively, to produce a 5' overhang. FokI is a bipartite endonuclease with separate recognition and cleavage domains. Because of its bipartite nature, FokI has received considerable interest in generating chimeric nucleases for use in biotechnology, and recently as possible therapeutic agents in gene therapy by initiating homologous gene recombination and repair. Here we show, using single-particle electron microscopic studies, that the FokI active complex prefers a single conformation in which the subunits are arranged in a doughnut shape complex with protein-protein and possibly protein-DNA interactions stabilizing the cleavage complex. Our electron microscopy (EM) model provides new insights into the activation mechanism of FokI and how non-specific cleavage is avoided.
[Show abstract][Hide abstract] ABSTRACT: Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded beta-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue non-native beta-hairpin. The structure we report has similarities to the hairpin formed by the A(beta)((1-40)) protofilament, yet also contains closely packed side-chains in a "steric zipper" arrangement found in the cross-beta spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.
Proceedings of the National Academy of Sciences 11/2006; 103(44):16248-53. DOI:10.1073/pnas.0607815103 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rotavirus double-layered particle (DLP) is a molecular machine that transcribes 11 genomic segments of double-stranded RNA into full-length mRNA segments during viral replication. DLPs from the human Wa strain of virus, belonging to subgroup II (SG II), possess a significantly reduced level of transcriptase activity compared to bovine UK DLPs that belong to subgroup I (SG I). Cryo-electron microscopy and icosahedral image analysis was used to define the structural basis for this difference in transcriptase activity and to derive three-dimensional density maps of bovine UK and human Wa DLPs at 26 angstroms and 28 angstroms resolution, respectively. The two rotavirus strains had the same diameter, T = 13 l icosahedral lattice symmetry and size of the VP6 trimers on the surface of the DLPs. However, the Wa particles displayed a remarkable absence of VP6 trimers surrounding each 5-fold vertex position. To further explore these structural differences, three-dimensional reconstructions were generated of DLPs decorated with Fab fragments derived from subgroup-specific monoclonal antibodies. The X-ray structures of VP6 and a generic Fab fragment were then docked into the cryo-electron microscopy density maps, which allowed us to propose at "pseudo-atomic" resolution the locations of the amino acid residues defining the subgroup-specific epitopes. Our results demonstrate a correlation between the structure of the VP6 layer and the transcriptase activity of the particles, and suggest that the stability of VP6 trimers, specifically those at the icosahedral 5-fold axes, may be critical for mRNA synthesis. Thus, subgroup specificity of rotavirus may reflect differences in the architecture of the double-layered particle, with resultant consequences for viral mRNA synthesis.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions. We show that the mature core contains 240 subunits in a T = 4 arrangement similar to that in expressed core (T is the triangulation number and the icosahedral shell contains 60 T subunits). During the infective cycle, the core assembles in an immature state around a complex of viral pregenomic RNA and polymerase. After reverse transcription with concomitant degradation of the RNA, the now mature core buds through a cellular membrane containing the surface proteins to become enveloped. Envelopment must not happen before reverse transcription is completed, so it has been hypothesized that a change in capsid structure may signal maturation. Our results show significant differences in structure between the RNA- and DNA-containing cores. One such difference is in a hydrophobic pocket, formed largely from residues that, on mutation, lead to abnormal secretion. We suggest that the changes we see are related to maturation and control of envelopment, and we propose a mechanism based on DNA synthesis for their triggering.
Proceedings of the National Academy of Sciences 12/2005; 102(44):15821-6. DOI:10.1073/pnas.0504874102 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Native fluorescence spectroscopy (NFS), primarily from tryptophan (trp), was used for in situ investigation of the virus-receptor attachment process in phi6, a lipid-containing bacteriophage from the Cystoviridae family. NFS allowed us to monitor the viral attachment directly to its receptor, which was isolated from the pseudomonad host. Immediately upon mixing, an increase in tryptophan emission intensity was observed followed by a subsequent decrease in emission intensity. The initial increase in emission intensity reflects changes in trp quantum efficiency as the phi6 surface proteins change their conformation as a result of virus attachment to the pilus. The cystovirus spike protein P3 is responsible for receptor recognition and the fluorescence changes observed are likely to be the consequence of its conformational transition at this initial infection stage, providing a kinetic view of this process. The subsequent decrease in trp emission intensity could be due to changes in viral proteins as a result of disassembly of the pili. The technique may have important applications for the dynamic monitoring of additional stages of the virus replication cycle such as assembly, interaction with nucleic acids and maturation. This work expands on a previous demonstration that fluorescence offered a novel tool to detect virus particle interaction with its host cell.
Photochemistry and Photobiology 07/2005; 81(4):879-83. DOI:10.1562/2005-01-14-RA-416 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.
[Show abstract][Hide abstract] ABSTRACT: Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.
Journal of Virology 01/2004; 77(24):13036-41. DOI:10.1128/JVI.77.24.13036-13041.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The WW domains are small proteins that contain a three-stranded, antiparallel beta-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electron-diffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-beta diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.
Proceedings of the National Academy of Sciences 09/2003; 100(17):9814-9. DOI:10.1073/pnas.1333907100 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimer's disease, Down's syndrome, and various dementing tauopathies. In Alzheimer's disease and Down's syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-beta structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-beta structure.
Proceedings of the National Academy of Sciences 08/2003; 100(15):9034-8. DOI:10.1073/pnas.1530287100 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.
Proceedings of the National Academy of Sciences 05/2002; 99(8):5591-5. DOI:10.1073/pnas.042681399 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The most common degenerative diseases of the human brain are characterized by the presence of abnormal filamentous inclusions in affected nerve cells and glial cells. These diseases can be grouped into two classes, based on the identity of the major proteinaceous components of the filamentous assemblies. The filaments are made of either the microtubule-associated protein tau or the protein alpha-synuclein. Importantly, the discovery of mutations in the tau gene in familial forms of frontotemporal dementia and of mutations in the alpha-synuclein gene in familial forms of Parkinson's disease has established that dysfunction of tau protein and alpha-synuclein can cause neurodegeneration.
Philosophical Transactions of The Royal Society B Biological Sciences 03/2001; 356(1406):213-27. DOI:10.1098/rstb.2000.0767 · 7.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Filamentous inclusions made of -synuclein constitute the defining neuropathological characteristic of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Rare familial cases of Parkinson's disease are associated with mutations A53T and A30P in -synuclein. We report here the assembly properties and secondary structure characteristics of recombinant -synuclein. Carboxy-terminally truncated human -synuclein (1-87) and (1-120) showed the fastest rates of assembly, followed by human A53T -synuclein,and rat and zebra finch alpha-synuclein. Wild-type human alpha-synuclein and the A30P mutant showed slower rates of assembly. Upon shaking, filaments formed within 48 h at 37°C. The related proteins beta- and gamma-synuclein only assembled after several weeks of incubation. Synthetic human alpha-synuclein filaments were decorated by an antibody directed against the carboxy-terminal 10 amino acids of alpha-synuclein, as were filaments extracted from dementia with Lewy bodies and multiple system atrophy brains. Circular dichroism spectroscopy indicated that alpha-synuclein undergoes a conformational change from random coil to beta-sheet structure during assembly. X-ray diffraction and electron diffraction of the alpha-synuclein assemblies showed a cross-beta conformation characteristic of amyloid.
Proceedings of the National Academy of Sciences 04/2000; 97(9):4897-4902. DOI:10.1073/pnas.97.9.4897 · 9.67 Impact Factor