Crystal structure of native chicken fibrinogen at 2.7 A resolution

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 A. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the alpha and beta chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The alphaC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.     

Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy.

We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane. This work shows that twin boundaries are highly structured regions that are directly related to the organization of units within each crystal domain. Projection maps from TEM images have shown that there are significant differences in the final average maps has allowed us to relate high magnification views obtained by AFM to the relatively high resolution information obtained by electron microscopy and image processing.     

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.     

Crystal structure of recombinant rabbit interferon-gamma at 2.7-A resolution

The crystal structure of recombinant rabbit interferon-gamma was solved by the multiple isomorphous replacement technique at 2.7-A resolution and refined to a crystallographic R-factor of 26.2%. The interferon crystallizes with one-half of the functional dimer in the asymmetric unit, with the two polypeptide chains of the dimer related by a crystallographic 2-fold symmetry axis. The structure is predominantly alpha-helical with extensive interdigitation of the alpha-helical segments of the two polypeptide chains.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Refined structure of desmodium yellow mottle tymovirus at 2.7 A resolution

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.     

Shell structure in Difflugia Lobostoma observed by scanning and transmission electron microscopy

Observations by scanning and transmission electron microscopy provide information about shells of Difflugia lobostoma which suggests a complex activity in shell construction. As observed by scanning microscopy, the shell consists of a single layer of sand grains which are organized into rosettes. The sand grains of the rosettes are different in size from those of flat areas between rosettes suggesting that the organism sorts these stones and places them according to size. Hydrofluoric acid treatment dissolves the sand but leaves a web of cement material intact. Examination of such acid treated specimens by transmission microscopy shows structure in the cement material of the shell, and granules of similar structure in the cell body. The rosette pattern observed differs from shell patterns in other species of Difflugia, and this suggests that shell structure may be species specific.     

Crystal structure of Streptomyces erythraeus trypsin at 2.7 A resolution.

The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 A resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4,968 reflections between 8 and 2.7 A resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Comparison of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 3(10)-helix is found through Ala56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.     

Low resolution structure of the influenza C glycoprotein determined by electron microscopy

The influenza C glycoprotein is clearly seen to be a trimer in specimens prepared with uranyl stains. Three-dimensional reconstructions from naturally occurring hexagonal arrays show that at low resolution (~30 Å) the influenza C glycoprotein exhibits similar features to the haemagglutinin glycoprotein of influenza A. Both have a triangular stalk near the membrane. Further from the membrane, the stalk becomes broader and the monomers more separated, leaving an open centre. The molecule narrows at the top. The regions of greatest contact between adjacent trimers in the arrays are situated nearer the distal end of the molecule. These contact zones can be related to equivalent zones on the influenza A haemagglutinin. Differences between the structure of the influenza A haemagglutinin glycoprotein determined by X-ray analysis and reconstructions of the influenza C glycoprotein are greatest at either end of the molecule, where the reconstructions are least reliable. Ordered glycoprotein arrays have not been observed on influenza C virions incubated at low pH. The staining patterns of glycoproteins on intact virions are essentially determined by the pH at which the virus is incubated, and the stain type, but not the pH of the stain.     

A cone formation theory of contamination in high resolution transmission electron microscopy

The formation theory of the contamination cone in high resolution transmission electron microscopy is discussed on the basis of a time dependent series of micrographs and their tilted images for evaluating the contamination process. For a high current density of an electron probe focused on the specimen, it was observed that two contaminated cones covering both upper and lower surfaces of a carbon substrate are formed as a function of the exposure time of the electron beam. From the measurement of the cross-section and height of contaminated cones, the contamination rate together with the volume rate, and time dependent shapes of various cones were analysed. The analysis was dependent on the diffusion cross-section and current density together with an average lifetime of adsorption molecules.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Crystal structure of human bone morphogenetic protein-2 at 2.7 A resolution

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta (TGF-beta) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecular replacement and refined to an R-value of 24.2 % at 2.7 A resolution. A common scaffold of BMP-2, BMP-7 and the TGF-betas, i.e. the cystine-knot motif and two finger-like double-stranded beta-sheets, can be superimposed with r. m.s. deviations of around 1 A. In contrast to the TGF-betas, the structure of BMP-2 shows differences in the flexibility of the N terminus and the orientation of the central alpha-helix as well as two external loops at the fingertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be specific for the respective BMP-subgroup. Two identical helix-finger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these specific features in the interaction of BMP-2 with its binding partners is discussed.     

The crystal structure of muscle phosphoglucomutase refined at 2.7-angstrom resolution.

A model of rabbit muscle phosphoglucomutase was refined at 2.7-A resolution by using two heavy atom derivatives for initial phasing and standard refinement procedures, including molecular replacement averaging about a 2-fold axis and dynamic simulation: final R-factor, 0.223 (no solvent modeling); RMS deviation from standard bond lengths and angles, 0.020 A and 3.6 degrees, respectively (all 8658 nonhydrogen atoms plus 36,953 reflections (F/sigma greater than or equal to 3) between 8- and 2.7-A resolutions); average of individually refined atomic B-factors, 40 A2 (all atoms) and 30 A2 (all atoms in domains I-III). An H-bonding scheme with 538 main chain H-bonds for the two monomers in the asymmetric unit and probable ligands for six uranyl ions in one heavy atom derivative is given. The monomer contains 42 strands/helices arranged into four alpha/beta-domains. Each of the first three domains contains an alpha 3 beta 4 alpha 1 motif, where the topology of beta 4 is 2,1,3,4:[arrows: see text] which is a topology not encountered in an extensive search among known protein structures. A spatial similarity is observed between corresponding residues in the three repetitions of this motif per monomer, but the minimal mutational distance between spatially corresponding residues is not statistically significant. The loop between the antiparallel strands in each of these domains is an important feature of the active site. In domain IV, beta-sheet topology is 2,1,3,4,5,6:[arrows:see text]. Noncovalent domain/domain interactions within the monomer are greatest between adjacent domains along the polypeptide chain, which are not substantially interdigitated and can be cleanly disengaged by altering the phi/psi torsional angles of three uniquely positioned residues in the model. The observed hierarchy of noncovalent interactions between structural units within the crystal, based on a semi-empirical paradigm, suggests that monomer-monomer contacts within the asymmetric unit are formed during growth of the lattice and provides a rationale for some of the diffraction characteristics of phosphoglucomutase crystals. An unusually deep crevice involving 58 residues is formed by the head-to-tail, twisted semicircular arrangement of the four domains of the monomer that places no atom more than 12 A from the water-accessible surface. The active site of the enzyme is extensively buried at the bottom of this crevice, at the approximate confluence of the four domains. Other features of the active site, including the surrounding helical dipoles, and the metal-ion binding pocket are described, together with structure/function comparisons with a number of other enzymes.     

Conventional transmission electron microscopy

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see

Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy.

Purple membranes adsorbed to mica were imaged in buffer solution using the atomic force microscope. The hexagonal diffraction patterns of topographs from the cytoplasmic and the extracellular surface showed a resolution of 0.7 and 1.2 nm, respectively. On the cytoplasmic surface, individual bacteriorhodopsin molecules consistently exhibited a distinct substructure. Depending on the pH value of the buffer solution, the height of the purple membranes decreased from 5.6 nm (pH 10.5) to 5.1 nm (pH 4). The results are discussed with respect to the structure determined by cryo-electron microscopy.     

Combining electron microscopy and comparative protein structure modeling

Recently, advances have been made in methods and applications that integrate electron microscopy density maps and comparative modeling to produce atomic structures of macromolecular assemblies. Electron microscopy can benefit from comparative modeling through the fitting of comparative models into electron microscopy density maps. Also, comparative modeling can benefit from electron microscopy through the use of intermediate-resolution density maps in fold recognition, template selection and sequence-structure alignment.     

Visualization of ceramide channels by transmission electron microscopy

Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.