Structure of Ca2+ release channel at 14 A resolution

Abstract

The 14 A resolution structure of the 2.3 MDa Ca2+ release channel (also known as RyR1) was determined by electron cryomicroscopy and single particle reconstruction. This structure was produced using collected data used for our previous published structures at 22-30 A resolution, but now taking advantage of recent algorithmic improvements in the EMAN software suite. This improved map clearly exhibits more structural detail and allows better defined docking of computationally predicted structural domain folds. Using sequence-based fold recognition, the N-terminal region of RyR1, residues 216-572, was predicted to have significant structural similarity with the IP3-binding core region of the type 1 IP3R. This putative structure was computationally localized to the clamp-shaped region of RyR1, which has been implicated to have a regulatory role in the channel activity.

Figure 1

Stereo views of the 3D structure of RyR1 at 14 Å resolution. The structure is shown at an oblique angle as viewed from the cytoplasm (a) and in a side view (b) with the cytoplasmic side facing upward. The reconstruction was generated using ~22,000 individual particle images collected on a JEOL 1200EX electron cryomicroscope, equipped with a tungsten filament and operated at 100 kV under minimal dose conditions (5–7 e/Ų) and at the defocus range of 0.9–4.1 μm. The closed channel conformation was obtained by the depletion of Ca²⁺ with 1 mM EGTA., The threshold level corresponds to an enclosed protein mass of 2.5 MDa assuming a protein density of 1.35 g/cm³. The scale bar represents 100 Å.

Figure 2

Fourier Shell Correlation (FSC) curve for the reconstruction as produced by the EMAN program eotest. To avoid any possibility of initial model bias, no reference was made to any previous model during refinement. The structure was refined from an initial model generated from automatically generated top and side views of the particle with 4-fold symmetry enforced. The overall refinement methodology was the same as a typical iterative EMAN refinement: projection-based particle classification: generation of a contrast transfer function (CTF)-corrected average for each class followed by reconstruction of a new 3D model from the class-averages. This refinement is iterated until convergence is achieved. The algorithm improvements which allowed this resolution improvement are described in detail elsewhere. First, the precision of the 2D alignment routine was improved, producing more accurate classifications. Second, classification accuracy was also enhanced through use of a new similarity criterion incorporating a true per-particle Wiener filter. Third, the resolution of the individual class-averages was improved through gradual reduction of the iterative self-alignment process used to produce class-averages. Resolution in the final reconstruction was determined using the standard technique of splitting the data into even and odd halves and calculating a FSC curve. In this case the most conservative, 0.5 threshold value was used. Since the determined 14 Å resolution is already approaching the traditional sampling limit of 3× oversampling, claiming any resolution beyond this point would not be justified.

Figure 3

Localization of the IP₃-like region of RyR1. (a) Homology model for the InP₃-like RyR1 domain (blue/red ribbon) and corresponding 14 Å density map (transparent red) generated using EMAN. The sequence corresponding to the LZ1 region residues 541–572 is shown with red ribbon. A homology model was generated by mapping of the coordinates of the X-ray structure of the IP₃-binding core region (1N4K) and the sequence residues 216–572 of RyR1 aligned to them. The model was further enhanced by the addition of side-chains using the SCWL algorithm. Sequence alignment, secondary structure prediction and homology modeling were performed using 3D-PSSM Fold Recognition server. (b) Fitting of 14 Å electron density map of the IP₃-like domain within RyR1 tetramer shown in tilted stereo top-view. Assignment of mass densities for the IP₃-like domain was performed using 6D fitting program Foldhunter as previously described. The top four correlation peaks with local correlation coefficients of about 0.91 (Foldhunter score) are found in the clamp-shaped domain of each of the four monomers in the RyR1 reconstruction. This fit had a z-score of 1.97. z-Score is a statistical measure of the number of standard deviations from the mean, which, in this case, corresponds a confidence level greater than 95%. A star marks the C terminus of the modeled region.