Biomedical subjects

Robert M Glaeser

Publications and source records attributed to Robert M Glaeser.

18 recordsLinked to original sources

PubMed2006

Design of a microfabricated, two-electrode phase-contrast element suitable for electron microscopy.

A miniature electrostatic element has been designed to selectively apply a 90 degrees phase shift to the unscattered beam in the back focal plane of the objective lens, in order to realize Zernike-type, in-focus phase contrast in an electron microscope. The design involves a cylindrically shaped, biased-voltage electrode, which is surrounded by a concentric grounded electrode. Electrostatic calculations have been used to determine that the fringing fields in the region of the scattered electron beams will cause a negligible phase shift as long as the ratio of electrode length to the transverse feature size is greater than 5:1. Unlike the planar, three-electrode einzel lens originally proposed by Boersch for the same purpose, this new design does not require insulating layers to separate the biased and grounded electrodes, and it can thus be produced by a very simple microfabrication process. Scanning electron microscope images confirm that mechanically robust devices with feature sizes of approximately 1 microm can be easily fabricated. Preliminary experimental images demonstrate that these devices do apply a 90 degrees phase shift between the scattered and unscattered electrons, as expected.

Journal Article↗

PubMed2006

Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin.

Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.

Electrons↗

PubMed2006

SPARX, a new environment for Cryo-EM image processing.

SPARX (single particle analysis for resolution extension) is a new image processing environment with a particular emphasis on transmission electron microscopy (TEM) structure determination. It includes a graphical user interface that provides a complete graphical programming environment with a novel data/process-flow infrastructure, an extensive library of Python scripts that perform specific TEM-related computational tasks, and a core library of fundamental C++ image processing functions. In addition, SPARX relies on the EMAN2 library and cctbx, the open-source computational crystallography library from PHENIX. The design of the system is such that future inclusion of other image processing libraries is a straightforward task. The SPARX infrastructure intelligently handles retention of intermediate values, even those inside programming structures such as loops and function calls. SPARX and all dependencies are free for academic use and available with complete source.

Computational Biology↗

PubMed2006

The parallelization of SPIDER on distributed-memory computers using MPI.

We describe the strategies and implementation details we employed to parallelize the SPIDER software package on distributed-memory parallel computers using the message passing interface (MPI). The MPI-enabled SPIDER preserves the interactive command line and batch interface used in the sequential version of SPIDER, thus does not require users to modify their existing batch programs. We show the excellent performance of the MPI-enabled SPIDER when it is used to perform multi-reference alignment and 3-D reconstruction operations on a number of different computing platforms. We point out some performance issues when the MPI-enabled SPIDER is used for a complete 3-D projection matching refinement run, and propose several ways to further improve the parallel performance of SPIDER on distributed-memory machines.

Algorithms↗

PubMed2006

Membrane-protein stability in a phospholipid-based crystallization medium.

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.

Bacterial Proteins↗

PubMed2005

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Aminopeptidases↗

PubMed2005

High-throughput film-densitometry: an efficient approach to generate large data sets.

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.

Automation↗

PubMed2004

Specificity of anion binding in the substrate pocket of bacteriorhodopsin.

The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff base binding site and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side chain of S85. On the basis of these X-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K(d), for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

Amino Acid Substitution↗

PubMed2004

Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump.

Structural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild-type bR could be a hydroxyl-ion pump. A position between the protonated Schiff base and residue 85 serves as an anion-binding site in the mutant protein, and hydroxyl ions should have access to this site during the O-intermediate of the wild-type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier.

Anion Transport Proteins↗

PubMed2004

Energy transduction in transmembrane ion pumps.

Recent crystallographic structures of three different ion pumps provide a first view of the mechanisms by which these molecular machines transfer ions across cell membranes against an electrochemical gradient. Each of the structures reinforces the concept that several buried counter ions have central roles in substrate recruitment, substrate binding and energy transduction during ion pumping. The spatial organization of the counter ions suggests that, initially, one or more counter ions lowers the Born energy cost of binding a substrate ion in the low-dielectric interior of the membrane. Subsequently, a ligand-induced conformational change seems to close a charged access gate to prevent backflow from a subsequent, low-affinity state of the pump. A final role of the buried counter ions might be to couple the input of external energy to a small charge separation between the substrate ion and the buried counter ions, thereby decreasing the binding affinity for the substrate ion in preparation for its release on the high-energy side of the membrane.

Animals↗

PubMed2004

Electron microscopy of biological macromolecules: bridging the gap between what physics allows and what we currently can get.

The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Because specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at 0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25%-35% of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.

Crystallization↗

PubMed2003

Crystal structure of the bromide-bound D85S mutant of bacteriorhodopsin: principles of ion pumping.

We report the crystal structure of a bromide-bound form of the D85S mutant of bacteriorhodopsin, bR(D85S), a protein that uses light energy rather than ATP to pump halide ions across the cell membrane. Comparison of the structure of the halide-bound and halide-free states reveals that both displacements of individual side-chain positions and concerted helical movements occur on the extracellular side of the protein. Analysis of these structural changes reveals how this ion pump first facilitates ion uptake deep within the cell membrane and then prevents the backward escape of ions later in the pumping cycle. Together with the information provided by structures of intermediate states in the bacteriorhodopsin photocycle, this study also suggests the overall design principles that are necessary for ion pumping.

Bacteriorhodopsins↗

PubMed2002

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy.

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.

Aminopeptidases↗

PubMed2002

Modeling chemical bonding effects for protein electron crystallography: the transferable fragmental electrostatic potential (TFESP) method.

This paper addresses the problem of determining the electrostatic potential of large proteins by the superposition of potentials calculated for small fragments. The use of different atomic and molecular fragments is considered for reproducing the molecular electrostatic potential of different conformations of N-acetylalanine methylamide (NAAMA) with an acceptable degree of error as measured by conventional R factors used in crystallographic structure refinement. Three different divisions of NAAMA are tested, producing fragments that incorporate increasingly more complete descriptions of molecular bonding with diminishing accuracy in geometric fit to the parent molecule: single atoms in molecules, bonded atoms in molecules and selected functional groups, such as the backbone peptide moiety, or the alpha-carbon, beta-carbon and their associated H atoms. In the resolution range 2.5-25A, the fairly straightforward use of single atoms in molecules reduces the calculated R factors by 5-15% over a free-atom superposition. No significant further improvement was found at the lowest resolutions with a superposition of single bonds in molecules and R factors were found to degrade with larger fragments at higher resolutions because of poor geometry fits to the atoms of the parent molecule. Because the potential distribution even for single atoms depends on the environment, the best accuracy will be obtained by using a library of fragment potentials calculated for each type of atom as a function of important protein conformations.

Algorithms↗

PubMed2002

Crystallization of membrane proteins from media composed of connected-bilayer gels.

The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.

Bacteriorhodopsins↗

PubMedDate not supplied

Historical background: Why is it important to improve automated particle selection methods?

A current trend in single-particle electron microscopy is to compute three-dimensional reconstructions with ever-increasing numbers of particles in the data sets. Since manual--or even semi-automated--selection of particles represents a major bottleneck when the data set exceeds several thousand particles, there is growing interest in developing automatic methods for selecting images of individual particles. Except in special cases, however, it has proven difficult to achieve the degree of efficiency and reliability that would make fully automated particle selection a useful tool. The simplest methods such as cross correlation (i.e., matched filtering) do not perform well enough to be used for fully automated particle selection. Geometric properties (area, perimeter-to-area ratio, etc.) and the integrated "mass" of candidate particles are additional factors that could improve automated particle selection if suitable methods of contouring particles could be developed. Another suggestion is that data be always collected as pairs of images, the first taken at low defocus (to capture information at the highest possible resolution) and the second at very high defocus (to improve the visibility of the particle). Finally, it is emphasized that well-annotated, open-access data sets need to be established in order to encourage the further development and validation of methods for automated particle selection.

Algorithms↗

PubMedDate not supplied

Automatic particle selection: results of a comparative study.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.

Algorithms↗

PubMedDate not supplied

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs.

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 A resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.

Algorithms↗