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Structures of well-folded RNA molecules can be determined with atomic resolution by either X-ray crystallography, cryo-EM, or NMR spectroscopy, but those of conformationally-flexible RNAs often are difficult to study with these methods. However, flexible RNAs have biological relevance and likely represent the majority of the RNA conformational space. Due to the high electron density of the phosphate-sugar backbone, RNA is very sensitive to small-angle X-ray scattering (SAXS), and SAXS data can be recorded with sub-μM concentrations and under near-physiological solution conditions without the need for labeling. For these reasons, SAXS has significant advantages over other techniques for obtaining global structural information of flexible RNAs in the form of molecular envelopes or low-resolution topological structural models. The SAXS-derived information is extremely valuable for bridging secondary structure data, often determined by other techniques, with a three-dimensional structure description. In this chapter, we present a detailed account of the principle, algorithms, and experimental and computational protocols for topological structure determination of RNA molecules in solution. To illustrate the applications of the methodology, we provide several case studies that cover a broad spectrum of the RNA conformational landscape.

Time-resolved structure determination of macromolecular conformations and ligand-bound intermediates is extremely challenging, particularly for RNA. With rapid technological advances in both microfluidic liquid injection and X-ray free electron lasers (XFEL), a new frontier has emerged in time-resolved crystallography whereby crystals can be mixed with ligand and then probed with X-rays (mix-and-inject) in real time and at room temperature. This chapter outlines the basic setup and procedures for mix-and-inject experiments for recording time-resolved crystallographic data of riboswitch RNA reaction states using serial femtosecond crystallography (SFX) and an XFEL.

The mechanism of a solid–solid phase transition in an RNA crystal is presented., Solid–solid phase transitions (SSPTs) occur between distinguishable crystalline forms. Because of their importance in application and theory in materials science and condensed-matter physics, SSPTs have been studied most extensively in metallic alloys, inorganic salts and small organic molecular crystals, but much less so in biomacromolecular crystals. In general, the mechanisms of SSPTs at the atomic and molecular levels are not well understood. Here, the ordered molecular rearrangements in biomacromolecular crystals of the adenine riboswitch aptamer are described using real-time serial crystallography and solution atomic force microscopy. Large, ligand-induced conformational changes drive the initial phase transition from the apo unit cell (AUC) to the trans unit cell 1 (TUC1). During this transition, coaxial stacking of P1 duplexes becomes the dominant packing interface, whereas P2–P2 interactions are almost completely disrupted, resulting in ‘floating’ layers of molecules. The coupling points in TUC1 and their local conformational flexibility allow the molecules to reorganize to achieve the more densely packed and energetically favorable bound unit cell (BUC). This study thus reveals the interplay between the conformational changes and the crystal phases – the underlying mechanism that drives the phase transition. Using polarized video microscopy to monitor SSPTs in small crystals at high ligand concentration, the time window during which the major conformational changes take place was identified, and the in crystallo kinetics have been simulated. Together, these results provide the spatiotemporal information necessary for informing time-resolved crystallography experiments. Moreover, this study illustrates a practical approach to characterization of SSPTs in transparent crystals.

Quorum sensing gene expression in vibrios is regulated by the LuxR/HapR family of transcriptional factors, which includes Vibrio vulnificus SmcR. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic but highly degenerate with sequence variations at each promoter. To examine the mechanism by which SmcR recognizes diverse DNA sites, we generated SmcR separation-of-function mutants that either repress or activate transcription but not both. SmcR N55I is restricted in recognition of single base-pair variations in DNA binding site sequences and thus is defective at transcription activation but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R and N142D substitutions disrupt the interaction with RNAP alpha but retain functional DNA binding activity. X-ray crystallography and small angle X-ray scattering data show that the SmcR DNA binding domain exists in two conformations (wide and narrow), and the protein complex forms a mixture of dimers and tetramers in solution. The three RNAP interaction-deficient variants also have two DNA binding domain conformations, whereas SmcR N55I exhibits only the wide conformation. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNAP and a multi-conformational DNA binding domain that permits recognition of variable DNA sites.

Solid–solid phase transitions (SSPTs) are widespread naturally occurring phenomena. Understanding the molecular mechanisms and kinetics of SSPTs in various crystalline materials, however, has been challenging due to technical limitations. In particular, SSPTs in biomacromolecular crystals, which may involve large-scale changes and particularly complex sets of interactions, are largely unexplored, yet may have important implications for time-resolved crystallography and for developing synthetic biomaterials. The adenine riboswitch (riboA) is an RNA control element that uses ligand-induced conformational changes to regulate gene expression. Crystals of riboA, upon the addition of a ligand, undergo an SSPT from monoclinic to triclinic to orthorhombic. Here, solution atomic force microscopy (AFM) and polarized video microscopy (PVM) are used to characterize the multiple transition states throughout the SSPT in both the forward and the reverse directions. This contribution describes detailed protocols for growing crystals directly on mica or glass surfaces for AFM and PVM characterization, respectively, as well as methods for image processing and phase-transition kinetics analysis.

Cobalamin riboswitch is a cis-regulatory element widely found in the 5'-UTRs of the vitamin B12-associated genes in bacteria, resulting in modulation and production of a particular protein. Thermoanaerobacter tengcongensis (Tte) AdoCbl riboswitches are the largest of the known riboswitches with 210 nucleotides, partially due to its long peripheral P6-extension, which enable high affinity of AdoCbl. Two structural elements, T-loop/T-looplike motif and kissing loop are key to the global folding of the RNA. While the structure of the TteAdoCbl riboswitch complex is known, we still do not understand the structure and conformation before AdoCbl ligand recognition. In order to delineate the conformational changes and the stabilities of long-range interactions, we have performed extensive all-atom replica-exchange molecular dynamics simulations of the TteAdoCbl riboswitch with a total simulation time of 2296 ns. We found that both the T-loop/T-looplike motif and kissing loop are very stable with ligand binding. The gating conformation changes of P6-extension allow the ligand to bind to the preorganized kissing loop binding pocket. The T-loop/T-looplike motif has much more hydrogen bonds than observed in TteAdoCbl riboswitch complex crystal structure, indicating an allosteric response of the T-loop/T-looplike motif. Our study demonstrated that the conformational ensemble of TteAdoCbl riboswitch provides stable structural elements for conformation selection and population shift in cobalamin recognition.

Nature Communications 12(1), 1762 (2021). doi:10.1038/s41467-021-21838-5, Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals., Published by Nature Publishing Group UK, [London]

HIV‐1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small‐angle X‐ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), (19)F site‐specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66(L289K). NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66(L289K) and p51 were predicted to form a heterodimer while p66 and p51(L289K) not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter‐subunit RNH‐thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Subdomain 5BSL3.2 of hepatitis C virus RNA lies at the core of a network of distal RNA–RNA contacts that connect the 5′ and 3′ regions of the viral genome and regulate the translation and replication stages of the viral cycle. Using small-angle X-ray scattering and NMR spectroscopy experiments, we have determined at low resolution the structural models of this subdomain and its distal complex with domain 3′X, located at the 3′-terminus of the viral RNA chain. 5BSL3.2 adopts a characteristic ‘L’ shape in solution, whereas the 5BSL3.2–3′X distal complex forms a highly unusual ‘Y’-shaped kissing junction that blocks the dimer linkage sequence of domain 3′X and promotes translation. The structure of this complex may impede an effective association of the viral polymerase with 5BSL3.2 and 3′X to start negative-strand RNA synthesis, contributing to explain the likely mechanism used by these sequences to regulate viral replication and translation. In addition, sequence and shape features of 5BSL3.2 are present in functional RNA motifs of flaviviruses, suggesting conserved regulatory processes within the Flaviviridae family.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription.

The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms., The centrosome is a membraneless organelle composed of two centrioles and an amorphous pericentriolar material but the overall centrosome organizations remains unknown. Here authors show that two scaffold proteins, Cep63 and Cep152, self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4.

Solid–solid phase transitions (SSPTs) have been widely observed in crystals of organic or inorganic small-molecules. Although SSPTs in macromolecular crystals have been reported, the majority involve local atomic changes, such as those induced by changes in hydration. SSPTs driven by large conformational changes, however, can be more difficult to characterize since they often significantly disrupt lattice packing interactions. Such drastic changes make the cooperativity of molecular motion at the atomic level less easily achieved and more dependent on intrinsic properties of the crystal that define lattice order. Here, we investigate the effect of crystal size on the uniformity of SSPT in thin plate-like crystals of the adenine riboswitch aptamer RNA (riboA) by monitoring changes in crystal birefringence upon the diffusion of adenine ligand. The birefringence intensity is directly related to molecular order and the concurrent changes to polarizability of molecules that results from structural changes throughout the phase transition. The riboA crystals were loosely grouped into three categories (small, medium, and large) based on the surface area of the crystal plates. The time width of transition increased as a function of crystal size, ranging from ∼13 s for small crystals to ∼40 s for the largest crystal. Whereas the transitions in small crystals (

Cobalamin riboswitch is a cis-regulatory element widely found in the 5’-UTRs of the vitamin B12-associated genes in bacteria, resulting in modulation and production of a particular protein. Thermoanaerobacter tengcongensis (Tte) AdoCbl riboswitches are the largest of the known riboswitches with 210 nucleotides, partially due to its long peripheral P6-extension, which enable high affinity of AdoCbl. Two structural elements, T-loop/T-looplike motif and kissing loop are key to the global folding of the RNA. While the structure of the TteAdoCbl riboswitch complex is known, we still do not understand the structure and conformation before AdoCbl ligand recognition. In order to delineate the conformational changes and the stabilities of long-range interactions, we have performed extensive all-atom replica-exchange molecular dynamics simulations of the TteAdoCbl riboswitch with a total simulation time of 2296 ns. We found that both the T-loop/T-looplike motif and kissing loop are very stable with ligand binding. The gating conformation changes of P6-extension allow the ligand to bind to the preorganized kissing loop binding pocket. The T-loop/T-looplike motif has much more hydrogen bonds than observed in TteAdoCbl riboswitch complex crystal structure, indicating an allosteric response of the T-loop/T-looplike motif. Our study demonstrated that the conformational ensemble of TteAdoCbl riboswitch provides stable structural elements for conformation selection and population shift in cobalamin recognition.

Effective gene regulation by the tetrahydrofolate riboswitch depends not only on ligand affinity but also on the kinetics of ligand association, which involves two cooperative binding sites. We have determined a 1.9-A resolution crystal structure of the ligand-free THF riboswitch aptamer. The pseudoknot binding site 'unwinds' in the absence of ligand, whereby the adjacent helical domains (P1, P2, and P3) become disjointed, resulting in rotation and misalignment of the gene-regulatory P1 helix with respect to P3. In contrast, the second binding site at the three-way junction, which is the first to fold, is structurally conserved between apo and holo forms. This suggests a kinetic role for this site, in which binding of the first ligand molecule to the stably folded three-way junction promotes formation of the regulatory pseudoknot site and subsequent binding of the second molecule. As such, these findings provide a molecular basis for both conformational switching and kinetic control.

It is considered that reserving a reasonable quantity of spare parts is an effective measure to increase equipment availability and reduce life cycle cost. Keeping the demand and configuration of equipment maintenance spare parts balanced is an important part to determine a reasonable amount of spare parts reserve. According to reliability theory and maintenance decision theory, under the assumed condition that the lifetime distribution and maintenance time distribution of weapon equipment are subject to exponential distribution, this paper concludes the decision models of two kinds of equipment maintenance spare parts, respectively repairable parts and irreparable parts.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a non-interacting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting Microcrystalline Tofacitinib Hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 A resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.

Structures of the four reaction states of the adenine riboswitch aptamer domain, including a transient intermediate state were solved by serial femtosecond crystallography. The structures not only demonstrate the use of X-ray free-electron lasers for RNA crystallography but have also proven that transient states can be determined in real time by mix-and-inject crystallography. These results illustrate the structural basis for the ligand-induced conformational changes associated with the molecular 'switch'.

In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA "closes" the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch-tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and antiterminator-acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.

Turn-on aptamers are in vitro-selected RNAs that bind to conditionally fluorescent small molecules and enhance their fluorescence. Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold). This aptamer was generated by structure-guided engineering and functional reselection of the parental aptamer Mango-III. Structures of both Mango-III and iMango-III have previously been determined by conventional cryocrystallography using synchrotron X-radiation. Using an X-ray free-electron laser (XFEL), the room-temperature iMango-III–TO1-biotin co-crystal structure has now been determined at 3.0 Å resolution. This structural model, which was refined against a data set of ∼1300 diffraction images (each from a single crystal), is largely consistent with the structures determined from single-crystal data sets collected at 100 K. This constitutes a technical benchmark on the way to XFEL pump–probe experiments on fluorescent RNA–small molecule complexes.

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 β-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent de novo SAD phasing. PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography.

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report co-crystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3’-end using an elaborate “discriminator” structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modelled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches, and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.