Summary About a decade ago, three electron cryomicroscopy (cryo-EM) single particle reconstructions of IP3R1 were reported at low resolution. Disturbingly, these structures bore little similarity to one another, even at the level of quaternary structure. Recently, we published an improved structure of IP3R1 at ~1 nm resolution. However, this structure did not bear any resemblance to any of the three previously published structures, leading to the question of why the new structure should be considered more reliable than the original three. Here we apply several methods, including class-average/map comparisons, tilt-pair validation, and use of multiple refinement software packages, to give strong evidence for the reliability of our recent structure. The map resolution and feature resolvability are assessed with the ‘gold standard’ criterion. This approach is generally applicable to assessing the validity of cryo-EM maps of other molecular machines.

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ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca2+ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N3ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC50 = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.

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The classification of knowledge of a specified subject area is an actual task. The well-known methods of obtaining decision trees using entropy are not suitable for the classification of the subject area knowledge. So, a new algorithm of obtaining decision trees, whose way of obtaining is approximated to the natural intelligence, is suggested in the article. Here, the knowledge of a subject area is presented as a complex of answers to questions, which help to find the solution to a current task. The connection of entropy with the appearance of knowledge, the classification of previous knowledge, and the definitions used in decision trees are also analyzed in the article. The latter is necessary to compare the suggested algorithm approximated to the natural intelligence with the traditional method, using a small example. The article contains the analysis of solving a classification task for such a subject area as optimization methods.

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