Michael Radermacher

Locations of Calmodulin and FK506-binding Protein on the Three-dimensional Architecture of the Skeletal Muscle Ryanodine Receptor

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large (≈80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.

Three-dimensional reconstruction from random projections: orientational alignment via Radon transforms

A method for alignment of projections with unknown projecting directions towards a three-dimensional reference has been developed. The technique has been applied to the three-dimensional reconstruction from images of a frozen-hydrated preparation of 50S ribosomal subunits from Escherichia coli recorded in the electron microscope. The algorithm as used here combines the Single Exposure Conical Reconstruction Technique (SECReT) with a three-dimensional orientation search. The algorithm allows for the refinement of a reconstruction obtained with SECReT by refinement of the true projection angles, and by the inclusion of projections with a priori unknown random orientation. With model data it is demonstrated that the algorithm works reliably even for signal-to-noise ratios lower than 1.

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three‐dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three‐dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.

Three-dimensional reconstruction of single particles negatively stained or in vitreous ice

The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.

Three-dimensional architecture of the skeletal muscle ryanodine receptor

Recent advances in determining the three‐dimensional architecture of the skeletal muscle ryanodine receptor/calcium release channel (RyR) by cryo‐electron microscopy and three‐dimensional reconstruction are discussed. The tetrameric receptor is characterized by a large 4‐fold symmetric cytoplasmic assembly that consists of many domains separated by solvent‐containing crevices and holes. Experimental evidence suggests that at least one regulatory ligand, calmodulin, binds to sites on the cytoplasmic assembly that are at least 10 nanometers from the transmembrane channel.

Structure of Ribosomes and Their Components by Advanced Techniques of Electron Microscopy and Computer Image Analysis

High-resolution electron microscopy plays a leading role in the structural analysis of biological macromolecules and is the most direct method for obtaining detailed information on the morphology, topography of the components, and functional sites of ribosomes. Electron microscopy (EM) has been important also for the interpretation of data obtained by a variety of physico-chemical techniques (for references see Chambliss et al., 1980; Liljas, 1982; Wittmann, 1983) on ribosomal protein locations, relative protein-protein distances, and protein-RNA binding sites, data which are more valuable when mapped within a well-defined structural framework provided, at present, by EM.

Functional Site Determinations in Three Dimensions on Eukaryotic and Eubacterial Ribosomes

Immuno electron microscopy has been a powerful tool in efforts to map exposures of individual r-proteins and functional sites on the ribosome, in that it has provided direct visual identification of the contact between an antibody and its epitope, as seen in an electron micrograph of a single-particle preparation of ribosomes. However, the information has been only two-dimensional: the site is seen on a projection image of the particle. If it can be identified on several different projections -- representing different orientations of the ribosome -- some three-dimensional (3D) information can be deduced, but the localization can be only approximate until 3D reconstruction techniques are applied to the problem.