Single-particle cryo-EM of calcium release channels: structural validation

Abstract

Few tools are available to determine the structure of large integral membrane proteins such as intracellular Ca(2+) release channels, RyRs and IP3Rs. Single particle cryo-EM can readily determine the structure of such channels to intermediate resolution, and can be used to quantitatively assess conformational variability. However, due to the, often low, image contrast of these cryospecimens, methods for validation are critical to insure the accuracy of such structures, and to put limits on their interpretability. The low-resolution structure of RyR has been well established for some time, but high-resolution has been slow to emerge. The structure of IP3R channel by cryo-EM had a number of false-starts, but improved validation methods have recently lead to a demonstrably accurate reconstruction.

Figure 1. Surface representation of 3D maps of Ca ²⁺ release channels determined by single-particle cryo-EM

(a, c) RyR1 (EMD-1275) [2]; (b, d) IP₃R1 (EMD-5278) [4]. (a, b) Structures of the tetrameric channels are viewed from the cytoplasmic side with one putative subunit is depicted in blue; (c, d) the two opposing subunits are viewed along membrane plane to allow visualization of internal features in the channel structure. The TM regions of RyR1 and IP₃R1 channels are quite similar, however the CY portions, aside from the difference in their overall dimensions, have quite different architectural arrangements including the most notable features such as a central plug in the IP₃R1 structure, and distinctive clamp-shaped regions in the RyR1 structure.

Figure 2. Validation of cryo-EM structures with different software packages

(a) 3D reconstructions of RyR1 (EMD-1275 [2] and EMD-5014) [3]) were filtered to the same ~12 Å resolution and rendered at a contour levels corresponding to a molecular mass of ~2.4 MDa. (b) 3D reconstructions of IP₃R1 were calculated in each software package using the same cryo-EM images [5], the final maps were filtered to match (~20 Å) and rendered at equivalent thresholds.

Figure 3. Tilt-pair analysis of images from vitrified IP₃R1

The tilt-pair parameter plots for 42 tilt pairs of particle images, recorded with a tilt angle of 10° in recently published study [5]. Surface representations of 3D maps used to determined orientations of particles from cryo-EM images: (a) EMDB-5278 [4]; (b) from [51]; (c) from [52]; (d) EMDB-1061 [53]. The plot in (a) clearly shows that the particles are clustered at the expected location based on the experimental tilt geometry, thus confirming veracity of cryo-EM map and its self-consistency [4]. The same tilt-pair validation failed with three cryo-EM structures (b–d) published a decade earlier thus demonstrating that these maps are in no way consistent with the current particle images.