3D Mapping of the SPRY2 Domain of Ryanodine Receptor 1 by Single-Particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform

In vivo confocal microscopy of the corneal endothelium: comparison of three morphometry methods after corneal transplantation

PURPOSE: The purpose of this study was to assess the endothelium of corneal grafts by in vivoconfocal microscopy (IVCM), and to evaluate an automated endothelial software system in comparison with a manual cell count and planimetry. PATIENTS AND METHODS: Overall, 40 corneal grafts (20 deep anterior lamellar keratoplasties (DALKs) and 20 penetrating keratoplasties (PKs)) were assessed by scanning-slit IVCM. The endothelial cell density (ECD) was estimated with the automated and the manual cell count method of the instrument's Nidek Advanced Vision Information System (NAVIS) software. The results were compared with planimetry as the reference method, and the agreement was assessed. RESULTS: The mean (±SD) automated ECD was 2278±524 cells/mm(2) (range 1167–3192 cells/mm(2)), whereas the manual cell count method gave significantly lower ECDs with a mean of 1213±677 cells/mm(2) (range 218–2440 cells/mm(2); P<0.001). The manual cell counts were also significantly lower than those by planimetry, with a mean ECD of 1617±813 cells/mm(2) (range 336–2941, P<0.001). Bland–Altman analyses indicated that the limits of agreement (LoA) between the automated and the planimetry method were −671 and +1992 cells/mm(2), whereas they were −1000 and +202 cells/mm(2) when comparing the manual cell counts with planimetry. CONCLUSION: Following keratoplasty, the NAVIS automated method is likely to overestimate endothelial cell counts due to oversegmenting of the cell domains. Automated ECDs are substantially higher than those by the manual counting method or planimetry. The differences are considerably larger post-keratoplasty than for normal corneas, and the methods should not be used interchangeably