Biomedical subjects

M van Heel

Publications and source records attributed to M van Heel.

At least 19 recordsLinked to original sources

PubMed1998

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Animals↗

PubMed1998

The 80S rat liver ribosome at 25 A resolution by electron cryomicroscopy and angular reconstitution.

BACKGROUND: The ribosome is central to protein synthesis in all living organisms. Single-particle electron cryomicroscopy has recently led to the determination of three-dimensional structures of bacterial ribosomes to approximately 20 A, which have since revolutionised our understanding of ribosomal function. The structure we present here of the 80S rat liver ribosome leads the way to similar progress for mammalian ribosomes. RESULTS: Among the new details revealed by our 25 A structure of the 80S rat liver ribosome are channels within the subunits, a large 'flat ribosomal surface' (FRS) on the outer surface of the large subunit and structural extensions of the mammalian compared to the bacterial ribosome. The main large subunit channel in both the bacterial and the mammalian species starts at the peptidyl transferase centre, below the central protuberance, and ends in the FRS, at the lower back of the large subunit. Structurally, the channels of both species can be directly superimposed. CONCLUSIONS: The mammalian structural extensions--none of which trespass the FRS--can be interpreted in terms of rRNA inserts and additional protein content over that of bacterial ribosomes. The main large subunit channel, which ends at the FRS, is the best candidate for the exit channel for proteins targeted for the endoplasmic reticulum.

Animals↗

PubMed1998

Multivariate analysis of single unit cells in electron crystallography.

High-resolution electron cryomicroscopy of two-dimensional protein crystals is associated with extremely noisy raw data in which even the crystal lattice often cannot be discerned. Correlation averaging procedures, aimed at calculating the total average of all unit cells of crystals in order to reduce noise, are now used routinely in electron crystallography. Multivariate statistical analysis (MSA) may be used for finding not only the average structure but also for quantifying the systematic departures from that average within the population of individual unit cells. We show that the MSA approach is applicable to single unit-cell images in the low-dose (< 10 electrons/A2), high-resolution (< 5 A) realm using 400 keV electron spot-scan images of ice-embedded gp32*I protein crystals. Our feasibility study opens a pathway toward exploiting these naturally occurring variations on the unit-cell theme in order to achieve higher-resolution three-dimensional reconstruction results, or to better understand the dynamic behaviour of molecules within two-dimensional crystals. We explain how single unit-cell images can be processed and classified into homogeneous groups, and we review how the results of such discriminate averaging may subsequently be exploited within the context of conventional "h, k"-space electron crystallographic approaches. Variations among the individual unit cells may thus be one of the most significant resolution-limiting factors currently experienced in electron crystallography. The quantitative assessment and exploitation of such variations may lead to an increased performance of electron crystallographic procedures.

Bacteriophage T4↗

PubMed1997

Visualization of elongation factor Tu on the Escherichia coli ribosome.

The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

Crystallography, X-Ray↗

PubMed1997

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre.

We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem-loop of the tRNA; there is no "allosteric" site. (2) The foot-print sites for P site tRNA that are essential for P site binding are similarly clustered around the P site anticodon stem-loop. The foot-print sites in 16 S rRNA helices 23 and 24 are, however, remote from the P site tRNA. (3) Cross-link sites from specific nucleotides within the anticodon loops of A or P site-bound tRNA are mostly in agreement with the model, whereas those from nucleotides in the elbow region of the tRNA (which also exhibit extensive cross-linking to the 50 S subunit) are more widely spread. Again, cross-links to helix 23 are remote from the tRNAs. (4) The corresponding cross-links from E site tRNA are predominantly in helix 23, and these agree with the model. Electron microscopy data are presented, suggestive of substantial conformational changes in this region of the ribosome. (5) Foot-prints for IF-3 in helices 23 and 24 are at a position with close contact to the 50 S subunit. (6) Foot-prints from IF-1 form a cluster around the anticodon stem-loop of A site tRNA, as do also the sites on 16 S rRNA that have been implicated in termination. (7) Foot-print sites and mutations relating to streptomycin form a compact group on one side of the A site anticodon loop, with the corresponding sites for spectinomycin on the other side. (8) Site-specific cross-links from mRNA (which were instrumental in constructing the 16 S model) fit well both in the upstream and downstream regions of the mRNA, and indicate that the incoming mRNA passes through the well-defined "hole" at the head-body junction of the 30 S subunit.

Base Sequence↗

PubMed1997

Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution.

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to approximately 45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the "functional units" within the dimeric "morphological units" of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

Animals↗

PubMed1997

Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy.

The three-dimensional structure of the translating 70S E. coli ribosome is presented in its two main conformations: the pretranslocational and the posttranslocational states. Using electron cryomicroscopy and angular reconstitution, structures at 20 A resolution were obtained, which, when compared with our earlier reconstruction of "empty" ribosomes, showed densities corresponding to tRNA molecules--at the P and E sites for posttranslocational ribosomes and at the A and P sites for pretranslocational ribosomes. The P-site tRNA lies directly above the bridge connecting the two ribosomal subunits, with the A-site tRNA fitted snugly against it at an angle of approximately 50 degrees, toward the L7/L12 side of the ribosome. The E-site tRNA appears to lie between the side lobe of the 30S subunit and the L1 protuberance.

Binding Sites↗

PubMed1996

Intrinsic versus imposed curvature in cyclical oligomers: the portal protein of bacteriophage SPP1.

Large cyclical oligomers may be formed by (curvi-) linear polymerization of monomers until the n(th) monomer locks in with the first member of the chain. The subunits in incomplete structures exhibit a natural curvature with respect to each other which can be perturbed when the oligomer closes cyclically. Using cryo-electron microscopy and multivariate statistical image processing we report herein a direct structural observation of this effect. A sub-population (approximately 15%) of incomplete oligomers was found within a sample of SPP1 bacteriophage portal proteins embedded in vitreous ice. Whereas the curvature between adjacent subunits of the closed circular 13-fold symmetric oligomer is 27.7 degrees, in these incomplete oligomers the angle is only 25.8 degrees, a value which almost allows for a 14-subunit cyclical arrangement. A simple model for the association of large cyclical oligomers is suggested by our data.

Microscopy, Electron↗

PubMed1996

Two structural configurations of the skeletal muscle calcium release channel.

Here we present the determination of the three-dimensional structure of the skeletal muscle Ca2+-release channel in an open state using electron cryomicroscopy and angular reconstitution. In contrast to our reconstruction of the channel in its closed state, the density map of the channel driven towards its open state, by the presence of Ca2+ and ryanodine, features a central opening in the transmembrane region-the likely passageway for Ca2+ ions from the sarcoplasmic reticulum to the cytosol. The opening of the channel is associated with a 4 degree rotation of its transmembrane region with respect to its cytoplasmic region, and with significant mass translocations within the entire cytoplasmic region of the channel tetramer.

Animals↗

PubMed1995

The 70S Escherichia coli ribosome at 23 A resolution: fitting the ribosomal RNA.

BACKGROUND: The ribosome--essential for protein synthesis in all organisms--has been an evasive target for structural studies. The best available structures for the 70S Escherichia coli ribosome or its 30S and 50S subunits are based on electron microscopical tilt experiments and are limited in resolution to 28-55 A. The angular reconstitution approach, which exploits the random orientations of particles within a vitreous ice matrix, can be used in conjunction with cryo-electron microscopy to yield a higher-resolution structure. RESULTS: Our 23 A resolution map of the 70S ribosome elucidates many structural details, such as an extensive system of channels within the 50S subunit and an intersubunit gap ideally shaped to accommodate two transfer RNA molecules. The resolution achieved is sufficient to allow the preliminary fitting of double-helical regions of an earlier three-dimensional ribosomal RNA model. CONCLUSIONS: Although we are still a long way from attaining an atomic-resolution structure of the ribosome, cryo-electron microscopy, in combination with angular reconstitution, is likely to yield three-dimensional maps with gradually increasing resolution. As exemplified by our current 23 A reconstruction, these maps will lead to progressive refinement of models of the ribosomal RNA.

Escherichia coli↗

PubMed1995

Electron cryomicroscopy and angular reconstitution used to visualize the skeletal muscle calcium release channel.

We exploit the random orientations of ice-embedded molecules imaged in an electron cryomicroscope to determine the three-dimensional structure of the Ca(2+)-release channel from the sarcoplasmic reticulum (SR) in its closed state, without tilting the specimen holder. Our new reconstruction approach includes an exhaustive search of all different characteristic projection images in the micrographs and the assignment of Euler angle orientations to these views. The 30 A map implied reveals a structure in which the transmembrane region exhibits no apparent opening on the SR lumen side. The extended cytoplasmic region has a hollow appearance and consists, in each monomer, of a clamp-shaped and a handle-shaped domain.

Animals↗

PubMed1994

Electron cryomicroscopy and digital image processing of lipoprotein(a).

Electron cryomicroscopy was used to study the structure of human lipoprotein(a) (Lp(a)), a plasma lipoprotein implicated in cardiovascular disease. An individual Lp(a) particle consists of a neutral lipid core within a shell of phospholipid, cholesterol and glycoprotein. In principle, electron cryomicroscopy images of single particles should contain structural detail attributable to the density differences among these components and the surrounding buffer. We observed such structural detail in images of frozen, hydrated Lp(a) particles. Lp(a) particles appeared to be roughly spherical in shape with an average diameter of 210 A. As is generally true for unstained samples in vitreous ice, imaged with a low electron dose, these images have low contrast with low signal-to-noise ratios. To increase the signal-to-noise ratio, we averaged classes of similar particles. We began with a set of 5813 randomly oriented Lp(a) particles and generated classes using a linear multivariate statistical method, followed by hierarchical ascendant classification. Our initial classification, based on only the first eight eigenvectors, separated particles on the basis of gross size and shape. After a rough reference-free alignment step, a second classification used the finer details in the images. This approach yielded class averages with structural detail only faintly visible in the raw, single images.

Freezing↗

PubMed1993

The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry.

Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail. From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.

Biological Transport↗

PubMed1991

A new family of powerful multivariate statistical sequence analysis techniques.

A novel multivariate statistical approach is presented for extracting and exploiting intrinsic information present in our ever-growing sequence data banks. The information extraction from the sequences avoids the pitfalls of intersequence alignment by analyzing secondary invariant functions derived from the sequences in the data bank rather than the sequences themselves. Such typical invariant function is a 20 x 20 histogram of occurrences of amino acid pairs in a given sequence or fragment thereof. To illustrate the potential of the approach an analysis of 10,000 protein sequences from the National Biomedical Research Foundation Protein Identification Resource is presented, whose analysis already reveals great biological detail. For example, zeta-hemoglobin is found to lie close to amphibian and fish chi-hemoglobin which, in turn, is an important clue to the physiological function of this mammalian early embryonic hemoglobin. The multivariate statistical framework presented unifies such apparently unrelated issues as phylogenetic comparisons between a set of sequences and distance matrices between the constituents of the biological sequences. The Multivariate Statistical Sequence Analysis (MSSA) principles can be used for a wide spectrum of sequence analysis problems such as: assignment of family memberships to new sequences, validation of new incoming sequences to be entered into the database, prediction of structure from sequence, discrimination of coding from non-coding DNA regions, and automatic generation of an atlas of protein or DNA sequences. The MSSA techniques represent a self-contained approach to learning continuously and automatically from the growing stream of new sequences. The MSSA approach is particularly likely to play a significant role in major sequencing efforts such as the human genome project.

Amino Acid Sequence↗

PubMed1990

Characteristic views of Escherichia coli RNA polymerase core enzyme in the scanning transmission electron microscope.

Eight characteristic views of Escherichia coli RNA polymerase core enzyme are presented which were found using multivariate statistical image processing of about 1000 individual molecular images. The data set was obtained from negatively stained specimens with a scanning transmission electron microscope (STEM) operated in dark field mode. The molecular projections were found to a resolution of around 30 A, thus visualizing new structural details of the core enzyme. An approximate twofold axis seems to relate the beta to the beta' subunit as well as both alpha units to each other.

DNA-Directed RNA Polymerases↗

PubMed1990

Fine structure of mitochondrial helical filaments revealed by computer image analyses.

In order to clarify the fine structure of the helical filaments appearing in the outer compartment of the mitochondria, Sprague-Dawley strain male rats were given 30% ethanol in drinking water for 90 days. The hepatic tissues of these animals were fixed with perfusion of glutaraldehyde via the portal vein followed by immersion in OsO4 and were then routinely processed for preparation of thin sections. Transmission electron micrographs of the sections were used for computer image analyses of the intramitochondrial helical filaments. With the application of the image analysis, it was revealed that the helical filaments show a right-hand rotating helix of 4.3 nm in thickness, 13.2 nm in diameter of the helix, and 15.7 nm in pitch. Also, with the multivariate statistical analysis and classification method of the IMAGIC image processing system, it was suggested in the classified images that the helical filament has substructures of rod-shaped particles of 4.3 nm in diameter. These particles are considered to be connected to each other and forming the helical structure of the filament.

Animals↗

PubMed1990

Comments on: 'Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli'.

In a recent paper Frank et al. (1988; J. Microsc. 150, 99-115) proposed the use of a hybrid clustering technique to sort macromolecular images. In contrast to the claims made in that paper, however, the proposed technique is less sophisticated than the enhanced hierarchical ascendant classification technique proposed by us a few years earlier. The hybrid approach will lead to partitions which are less optimal in terms of minimizing the total intra-class variance for a predetermined number of classes, irrespective of whether the data show distinct clusters or, rather, possess a quasi-continuous intrinsic structure.

Algorithms↗

PubMed1989

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy.

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

Bacterial Outer Membrane Proteins↗