Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle.

Abstract

Abstract. The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 ° rotation about an axis in the plane of the specimen grid.The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca2+-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.I N striated muscle the calcium release channel (CRC) ~, also known as the ryanodine receptor, plays a key role in excitation-contraction coupling, the process by which neuronal-induced depolarization of the sarcolemma leads to release of calcium from the lumen of the sarcoplasmic reticulum (SR). The CRC is an intracellular integral membrane protein of the SR (for reviews see Fleischer and Inui, 1989;McPherson and Campbell, 1993). Depolarization of the sarcolemma causes the CRC to open by mechanisms (apparently different in cardiac and skeletal muscle) whose elucidation represents the central remaining problem in understanding excitation-contraction coupling.Several groups succeeded in purifying the CRC from skeletal muscle, and in reconstituting Ca 2+ channel activity in lipid bilayers (Inui et al

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…Most cryo-EM studies on RyRs, (Radermacher et al 1992;Radermacher et al 1994;Serysheva et al 1995;Orlova et al 1996;Sharma et al 1998;Serysheva et al 1999;Benacquista et al 2000;Sharma et al 2000;Ludtke et al 2005;Samsó et al 2005;Serysheva et al 2005;Serysheva et al 2008;Samsó et al 2009) and all the subnanometer resolution analysis (Serysheva et al 2008;Samsó et al 2009) have focused on the RyR1, however, some progress has been made with RyR2 (Sharma et al 1998;Liu et al 2002) and RyR3 Liu et al 2001). Overall, the structures of all three isoforms are similar, consistent with the high sequence homology ( 65%).…”

Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel

“…Four residues (E161, R164, R402, and I404) would be Subregions are shown within one of the RyR1 subunits. The numbering of subregions is adopted according to a previous convention except with finer divisions (43). A subregion is interpreted as compact protein density, and the boundary of subregion tends to be weakly connected with its adjacent densities.…”

Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

“…Functional RyR molecules are homotetramers of 2.3 MDa (565 kDa per monomer), making RyRs the largest ion channels known to date and contributing to the difficulty in obtaining crystals suitable for atomic structure determination by x-ray crystallography. In the past decades, several groups have determined three-dimensional reconstructions of intact RyRs at intermediate resolutions by cryo-electron microscopy (cryo-EM) (7)(8)(9)(10)(11)(12). Recently, crystal structures of RyR1 fragments, mainly from the N-terminal region, have been solved to atomic resolution (13)(14)(15)(16).…”