RNA target highlights in CASP15: Evaluation of predicted models by structure providers

Abstract

The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x-ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA-protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.

Keywords: CASP; RNA folding; RNA structure prediction; community-wide experiment; cryo-EM; x-ray crystallography.

Figure 1:

(A) Secondary structure of the HDV ribozyme as deduced from the crystal structure (PDB: 1DRZ). Among the characteristic structural elements, P1 forms a nested double pseudoknot together with P1.1 and P2. (B) The secondary structure of the human and chimpanzee CPEB3 ribozymes as deduced from the crystal structures (C) show that the P1.1 element (purple) is not formed and instead the ribozymes form dimers with a neighboring molecule (gray shaded for clarity). The residues are numbered according to the P4 wild type sequence framed. (D-G) Comparison of representative models with the crystal structure of the human CPEB3 ribozyme. (D) In the crystal structure of the human CPEB3 ribozyme (PDB: 7QR4), the distal location of the P1.1 forming elements (purple) is indicated by a symbol (∣---∣).(E) R1107TS232_1, AIchemy_RNA2, RMSD 4.52 Å; (F) R1107TS054_3, Ultrafold, RMSD 8.13 Å; (G) R1107TS229_1, Yang_server, RMSD 17.92 Å.

Figure 2:

Covariation model and comparison of the preQ1-IIII riboswitch co-crystal structure to the best predicted CASP15 model. (A) Covariation model based on previous data. (B) Global superposition of the experimental model (PDB:8FZA, purple) with the top prediction model (R1117TS287_4, orange). (C) Close-up view of the preQ₁ binding pocket. The metabolite (green) was derived from the co-crystal structure. (D) Close-up of the pocket ceiling. (E) The expression platform showing WC pairing of Gua29 and Gua30 of the Shine-Dalgarno sequence.

Figure 3:

Comparing ribbon models of the experimentally determined structures (A, G, H) to the best predictions from the top 5 groups for CASP:R1128 (A-F, left) and CASP:R1138 (G-M, right). (B) R1128TS232 AIchemy_RNA2, (C) R1128TS287 Chen, (D) R1128TS147 SHT, (E) R1128TS227 GinobiFold, (F) R1127TS125 UltraFold_Server, (I) R1138TS232 AIchemy_RNA2, (J) R1138TS287 Chen, (K) R1138TS081 RNApolis, (L) R1138TS128 GeneSilico, (M) R1138TS227 GinobiFold.

Figure 4:

Categorization of all R1149 (SARS-CoV-2 SL5 domain) submitted models (A) and all R1156 (BtCoV-HKU5 SL5 domain) submitted models (C) by features they correctly predict. In the Venn diagram (not to scale), areas are labeled with the number of models that correctly predict the features whose circles overlaps in that area: base-pairing (blue) and base-stacking (green) at junction, angle between SL5-stem-SL5c and SL5a-SL5b (pink), and presence of SL5a-SL5c interaction (yellow, R1156 only). Angle between SL5-stem-SL5c and SL5a-SL5b for R1149 (B) and R1156 (D) colored by categories in (A) and (C) with the experimental structure range marked in pink; 0° is parallel orientation, 180° is an antiparallel, direction of rotation is defined from the view of F,H as moving SL5b clockwise. (E) For R1156 models, the bend angle of SL5a at the internal loop as measured by the angle between residues 24-27, 64-95 and residues 28-59. Three example models for R1149 (F-G) and R1156 (H-J) with SL5-stem in gray, SL5a in blue, SL5b in orange, and SL5c in red. (F,H) The predicted structures (dark) and the cryo-EM models (translucent) and GDT-TS score with rank over all models. (G,I) The predicted model’s 4-way junction with arrows showing 5’ to 3’ direction, and (J) the SL5a-SL5c interaction.

Figure 5:

(A) Summary of the overall average RMSD of all predictions in each of the 12 RNA targets. (B) The experimental secondary structure of RsmZ with protein binding site GGA marked in orange. (C) The secondary structure of RsmZ predicted by Yang-server with protein binding site GGA marked in orange. (D) Cryo-EM model of RsmZ colored the same as the secondary structure. (E) Predicted model of RsmZ by Yang-server colored the same as the secondary structure. (F) Superposition of the cryo-EM (gray) and Yang-server predicted (green) RsmZ structures aligned on the stacked SL1-SL2. (G) Superposition of the cryo-EM (gray) and Yang-server predicted (green) RsmZ structures aligned on the longest SLter. (H) Secondary structure of top-ranked models by TM-sore and GDT_TS of target R1189. (I) Superposition of all top-ranked models by TM-score and GDT_TS of target R1189 aligned on the protein binding site SL2 and SL3, with cyan from Venclovas, orange from CoDock, magenta from Kiharalab_Server. (J) Superposition of the RsmZ cryo-EM structure (gray) and a representative RsmZ model predicted by RNApolis (blue) aligned on SL2 and SL3.