From static structures to dynamic landscapes: cryo-EM redefines RNA biology

Reviewer #1 (Public review):

Summary:

The review addresses an important and timely topic that concerns the role of cryo-EM in transforming RNA structural biology from a "static" discipline to one increasingly concerned with conformational ensembles and molecular dynamics. The scope is well within the eLife standards, and the style and general architecture do fit eLife.

Strengths:

The review is extremely well written, well-conceived and clear. The main strengths are in the breadth of coverage, the clear theme, and the inclusion of practical examples that explain in detail the construct design, sample preparation, vitrification, and data analysis. The manuscript will certainly be impactful and valuable, especially for readers who are not specialists in cryo-EM, as it provides an accessible overview of recent advances across a wide range of RNA systems.

Weaknesses:

My only reservation is that, currently, the review reads too much like a list of examples. The authors should make an effort, and I am sure they are well up to it, to try to synthesise the message, provide more critical insights and amalgamate the text better, to really reach a wider audience.

If revised along the lines detailed below, I am sure that the review will become an authoritative and influential resource for the RNA structural biology community.

Reviewer #2 (Public review):

Summary:

In this review, the authors set out to synthesize how cryo-EM is reshaping RNA structural biology, moving the field from the determination of static, individual conformations toward the reconstruction of dynamic conformational ensembles and energy landscapes. Through eight case studies spanning ribozymes, riboswitches, viral RNAs, and synthetic RNA assemblies, they aim to show how cryo-EM has revealed mechanisms of RNA motion, including folding, ligand-dependent switching, and cooperative assembly, and to provide a practical account of the experimental and computational challenges (construct design, sample preparation, vitrification, data analysis) that are specific to dynamic RNA targets.

Strengths:

The manuscript succeeds in bringing together a wide and genuinely current range of case studies illustrating the field's shift toward dynamics-focused cryo-EM, several published within the last one to two years. The dedicated "Challenges" section, which walks through construct engineering, buffer and vitrification optimization, grid screening, and heterogeneity-resolving computational approaches, is a particularly useful and practical contribution; it goes beyond simply cataloguing structures and gives readers new to the area a genuine methodological roadmap. The figures are detailed and well matched to the quantitative claims made in the text (helix rotations, distances, RMSDs), which strengthens the paper's value as a reference resource.

Weaknesses:

The coverage of two areas in particular, the SL5 viral RNA element and RNA quaternary/multimeric assemblies, would benefit from incorporating additional recent primary literature that is directly relevant but currently omitted. This does not undermine the manuscript's core narrative, but it means the review is presently less complete than it could be as a field synthesis, particularly for readers using it to identify the full body of recent work on these specific RNA classes.

More concerning is that one specific structural claim, describing conformer heterogeneity in the cobalamin riboswitch (Case study 3, holo dimer 4), appears to invert the finding reported in its own source paper (Ding, Deme et al., 2023). As written, the manuscript states that P2 and the distal half of P6 are structured in dimer 4, whereas the source paper reports that these are the regions that could not be modeled. This is worth flagging prominently because it is a factual claim about a specific structure, not an interpretive point, and readers relying on this review as a secondary source could come away with an inverted understanding of that structure's flexibility.

The manuscript also contains several minor internal inconsistencies. None of these individually threatens the paper's core arguments, but together they suggest the manuscript would benefit from a careful proofreading and reference-list audit pass.

Overall, the authors largely achieve their stated aim. The case studies convincingly illustrate that cryo-EM can now resolve discrete and continuous conformational states of RNA at near-atomic resolution, and the Challenges section substantiates the claim that construct design, sample preparation, and computational innovations have been jointly responsible for this progress. The gaps in coverage of SL5 and multimeric RNA literature, and the inverted claim in Case study 3, are the main respects in which the manuscript falls short of being a fully comprehensive and accurate synthesis at this stage, but these are correctable issues rather than flaws in the overall argument or framework.

This review is likely to be a useful entry point and practical reference for researchers moving into RNA cryo-EM, particularly given the level of methodological detail in the Challenges section. Its impact would be strengthened by two additions. First, a short discussion of recent cryo-EM advances in tRNA would round out the manuscript's coverage of classical RNA structural targets alongside the ribozyme, riboswitch, and viral RNA case studies already included. Second, the raiA non-coding RNA, currently mentioned only briefly, has in the last two years become a genuine model system for cryo-EM-based ncRNA structure determination, progressing from a single novel-fold discovery to a comparative structural framework spanning multiple raiA subtypes and candidate protein partners. Expanding this into a full case study would let the manuscript showcase, in a single worked example, exactly the kind of field-level progression (from novel fold to scaffold-based strategy and further to comparative structural framework) that the review's own framing describes as the trajectory of the field as a whole.

Reviewer #3 (Public review):

This review describes how cryo-EM is moving RNA structural biology from the determination of static structures toward the characterization of conformational ensembles. Eight case studies spanning self-splicing introns, riboswitches, viral RNA elements and synthetic assemblies show how cryo-EM has captured folding intermediates, hinge-mediated domain motions and ligand-dependent switching, and the authors pair these examples with practical guidance on construct design, sample preparation, vitrification and heterogeneity analysis. The argument that conformational heterogeneity is a source of mechanistic information, rather than a limitation, is well supported, and Table 1 will be a useful reference for laboratories entering the field. The manuscript is timely and of broad interest, and I recommend publication after minor revision.

(1) Scaffold-based structure determination (page 14, lines 5-9). The section on chimeric RNAs presents the scaffold strategies as a single group and cites Haack et al. (2025) and Langeberg & Kieft (2023) in one parenthetical, so individual results are not attributed to their sources. The distinction between these studies is substantive. Earlier scaffolds based on the Tetrahymena group I intron (Langeberg & Kieft, 2023) or on RNA origami (Sampedro Vallina et al., Nucleic Acids Res. 51:4613-4624, 2023) resolved the appended RNAs at approximately 4.4-5 Å. Haack et al. (2025) reported the first RNA scaffold to yield a high-resolution structure of the target itself, resolving the ligand-binding pocket of the thiamine pyrophosphate (TPP) riboswitch at 2.5 Å and extending nucleotide-level cryo-EM analysis to small RNAs that had previously been intractable. I ask the authors to attribute each result to its source and to state explicitly that Haack et al. (2025) achieved the first high-resolution structure of a scaffolded target RNA. Because the same study captured the ligand-free TPP riboswitch in an open, Y-shaped conformation, a direct example of the ligand-dependent switching that is central to this review, the TPP riboswitch should also appear in the riboswitch section (page 8, lines 15-36), together with the fluoride riboswitch of Langeberg & Kieft (2023).

(2) Page 6, lines 8-14. Self-splicing introns are described collectively as evolutionary ancestors of the spliceosome that reside in pre-mRNA transcripts. The proposed ancestral relationship to spliceosomal introns and snRNAs applies to group II introns. Group I introns, including the Tetrahymena intron, which interrupts a pre-rRNA, initiate splicing with an exogenous guanosine and are not considered spliceosomal precursors.