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Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards , building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here. Introduction When the Protein Data Bank (PDB) (Protein Data Bank, 1971) was first established in 1971, X-ray crystallography was the only method for determining three-dimensional structures of biological macromolecules at sufficient resolution to build atomic models. A decade later, structures of biomolecules in solution could also be determined by nuclear magnetic resonance (NMR) spectroscopy (Williamson et al., 1985). Recently, three-dimensional cryoelectron microscopy (3DEM) (Henderson et al., 1990) began to achieve unprecedented near-atomic resolution for large complex assemblies. Increasingly, investigators are also modeling structures based on data from more than one method (Rout and Sali, 2019). These integrative/hybrid approaches to structure determination consist of collecting information about a system using multiple experimental and computational methods, followed by integrative/hybrid modeling that converts this information into integrative/hybrid structure models. For succinctness, we will use the term integra-tive hereafter to refer to integrative/hybrid approaches, modeling, and models.

Single-molecule Forster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between +/- 0.02 and +/- 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods. This work was supported by the European Research Council (ERC; grant agreement nos. 261227 (to A.N.K.), 646451 (to E.L.), 638536 (to T.C.), 671208 (to C.A.M.S.), and 681891 (to T. Hugel)), the Deutsche Forschungsgemeinschaft (DFG) (grant MI 749/4-1 to J.M., grant TI 329/10-1 to P.T., and grant SCHL 1896/3-1 to M.S.), the Swiss National Science Foundation (to B.S.), the German Federal Ministry of Education and Research (BMBF; 03Z2EN11 to M.S.), Research Foundation Flanders (FWO; grant G0B4915N to J. Hendrix), the Agency for Innovation by Science and Technology (IWT Flanders; doctoral scholarship to N.V.), the Danish Council for Independent Research (Sapere Aude grant 0602-01670B to V.B.), the Novo Nordisk Foundation (NNF15OC0017956 to V.B.), the UK BBSRC (grant BB/H01795X/1 to A.N.K.),the National Institute of Mental Health (grant MH081923 to M.E.B.), Clemson University (start-up funds to H. Sanabria, S.R.A., and I.S.Y.-O.), the NIH (grants GM109832 and GM118508 to K.R.W.; grant GM112659 to T. Ha), the NSF (Career Award MCS1749778 to H. Sanabria), the Carl-Zeiss-Stiftung (doctoral fellowship to E.K.), the Stipendienstiftung Rheinland-Pfalz (doctoral scholarship to G.K.), the Braunschweig International Graduate School of Metrology (B-IGSM; to B.W.), the DFG Research Training Group (GrK1952/1 "Metrology for Complex Nanosystems" to B.W.), the University of Sheffield (start-up funds to T.D.C.), and the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017R1A2B3010309 to N.K.L.).

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Riboswitches are noncoding regulatory elements that control gene expression in response to the presence of metabolites, which bind to the aptamer domain. Metabolite binding appears to occur through a combination of conformational selection and induced fit mechanism. This demands to characterize the structural dynamics of the apo state of aptamer domains. In principle, molecular dynamics (MD) simulations can give insights at the atomistic level into the dynamics of the aptamer domain. However, it is unclear to what extent contemporary force fields can bias such insights. Here, we show that the Amber force field ff99 yields the best agreement with detailed experimental observations on differences in the structural dynamics of wild type and mutant aptamer domains of the guanine-sensing riboswitch (Gsw), including a pronounced influence of Mg2+. In contrast, applying ff99 with parmbsc0 and parmχOL modifications (denoted ff10) results in strongly damped motions and overly stable tertiary loop-loop interactions. These results are based on 58 MD simulations with an aggregate simulation time11 μs, careful modeling of Mg2+ ions, and thorough statistical testing. Our results suggest that the moderate stabilization of the χ-anti region in ff10 can have an unwanted damping effect on functionally relevant structural dynamics of marginally stable RNA systems. This suggestion is supported by crystal structure analyses of Gsw aptamer domains that reveal χ torsions with high-anti values in the most mobile regions. We expect that future RNA force field development will benefit from considering marginally stable RNA systems and optimization toward good representations of dynamics in addition to structural characteristics.

Riboswitches are noncoding regulatory elements that control gene expression in response to the presence of metabolites, which bind to the aptamer domain. Metabolite binding appears to occur through a combination of conformational selection and induced fit mechanism. This demands to characterize the structural dynamics of the apo state of aptamer domains. In principle, molecular dynamics (MD) simulations can give insights at the atomistic level into the dynamics of the aptamer domain. However, it is unclear to what extent contemporary force fields can bias such insights. Here, we show that the Amber force field ff99 yields the best agreement with detailed experimental observations on differences in the structural dynamics of wild type and mutant aptamer domains of the guanine-sensing riboswitch (Gsw), including a pronounced influence of Mg 2 + . In contrast, applying ff99 with parmbsc0 and parmχ OL modifications (denoted ff10) results in strongly damped motions and overly stable tertiary loop–loop interactions. These results are based on 58 MD simulations with an aggregate simulation time > 11 μs, careful modeling of Mg 2 + ions, and thorough statistical testing. Our results suggest that the moderate stabilization of the χ - anti region in ff10 can have an unwanted damping effect on functionally relevant structural dynamics of marginally stable RNA systems. This suggestion is supported by crystal structure analyses of Gsw aptamer domains that reveal χ torsions with high- anti values in the most mobile regions. We expect that future RNA force field development will benefit from considering marginally stable RNA systems and optimization toward good representations of dynamics in addition to structural characteristics.

Riboswitches are genetic regulatory elements that control gene expression depending on ligand binding. The guanine-sensing riboswitch (Gsw) binds ligands at a three-way junction formed by paired regions P1, P2, and P3. Loops L2 and L3 cap the P2 and P3 helices and form tertiary interactions. Part of P1 belongs to the switching sequence dictating the fate of the mRNA. Previous studies revealed an intricate relationship between ligand binding and presence of the tertiary interactions, and between ligand binding and influence on the P1 region. However, no information is available on the interplay among these three main regions in Gsw. Here we show that stabilization of the L2-L3 region by tertiary interactions, and the ligand binding site by ligand binding, cooperatively influences the structural stability of terminal base pairs in the P1 region in the presence of Mg2+ ions. The results are based on molecular dynamics simulations with an aggregate simulation time of ~10 μs across multiple systems of the unbound state of the Gsw aptamer and a G37A/C61U mutant, and rigidity analyses. The results could explain why the three-way junction is a central structural element also in other riboswitches and how the cooperative effect could become contextual with respect to intracellular Mg2+ concentration. The results suggest that the transmission of allosteric information to P1 can be entropy-dominated.

The mechanical heterogeneity of biomolecular structures is intimately linked to their diverse biological functions. Applying rigidity theory to biomolecules identifies this heterogeneous composition of flexible and rigid regions, which can aid in the understanding of biomolecular stability and long-ranged information transfer through biomolecules, and yield valuable information for rational drug design and protein engineering. We review fundamental concepts in rigidity theory, ways to represent biomolecules as constraint networks, and methodological and algorithmic developments for analyzing such networks and linking the results to biomolecular function. Software packages for performing rigidity analyses on biomolecules in an efficient, automated way are described, as are rigidity analyses on biomolecules including the ribosome, viruses, or transmembrane proteins. The analyses address questions of allosteric mechanisms, mutation effects on (thermo-)stability, protein (un-)folding, and coarse-graining of biomolecules. We advocate that the application of rigidity theory to biomolecules has matured in such a way that it could be broadly applied as a computational biophysical method to scrutinize biomolecular function from a structure-based point of view and to complement approaches focused on biomolecular dynamics. We discuss possibilities to improve constraint network representations and to perform large-scale and prospective studies. WIREs Comput Mol Sci 2017, 7:e1311. doi: 10.1002/wcms.1311 For further resources related to this article, please visit the WIREs website.

Non-protein coding RNAs perform essential functions in living organisms. They commonly exist as dynamic ensembles of conformational states. While many structurally known RNAs are trapped in one or a few conformations by interactions with proteins or tertiary contacts between stems, bulges, or loops the knowledge of equilibrium structures, conformational space, and tertiary conformational changes of large RNAs not being restrained by external or tertiary interactions is still very limited.Helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Here we explore the topology of a set of a 4WJ and related 3WJs related to the hairpin ribozyme by measuring more than 250 different FRET-pairs using single-molecule multi-parameter fluorescence detection [1]. Using FRET restrained high-precision structural modeling combined with full atom MD simulations as a hybrid tool [2,3], we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ. By a suitable choice of the number of bases in the bulges the helices arrangements of the corresponding 3WJs can span a huge conformational space which is necessary for the stem communication in functional RNAs.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010).[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011).[3] Kalinin, S. et al. Nat. Methods 9, 1218-1225 (2012)

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing biomolecular structures. Here we apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) with a high level of precision in distance better than 1% of the Forster radius [1].We have generated a database of RNA 4WJs and six different RNA 3WJs with different bulges and sequences to study the influence of these factors on the junction conformations of RNA 3WJs. Overall 260 FRET pairs were measured with single-molecule MFD at 20 mM MgCl2 concentration and analyzed with the analysis toolkit [2] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.Monte Carlo simulations showed that sterically allowed conformational space for RNA junctions is large. However, FRET measurements detect the existence of three different static conformers for RNA 4WJ, whereas RNA 3WJs have only one predominant static conformation. terically allowed conformational space for RNA is large. Their junction geometry was described in terms of mutual and Euler angles between helices. The FRET-derived structures suggest that the sequence dictates a junction specific conformation within the large topology space. Furthermore we see that bulges in the junction region determine orientation and rotation of helices and induce coaxial stacking between two of them.[1] Antonik, M., et al., J.Phys.Chem.B, 110, 6970-6978 (2006)[2] Kalinin, S. et al, Nat. Meth., 9, 1218-1225 (2012)

Like for many other non-coding RNAs, helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Using FRET restrained high-precision structural modeling as a hybrid tool we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ based on the hairpin ribozyme. 51 different FRET-pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). For each dataset, the single-molecule approach allowed for the simultaneous extraction of three distances (and their corresponding errors) belonging to one major FRET state and two minor states. Distinct Mg2+-affinities were used for the assignment of the two minor states to the corresponding conformers. Rigid body models for the major and both minor conformers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by all-atom MD simulations and coarse-grained RNA folding using FRET-restraints. A cluster analysis gives confidence levels for the proposed ensemble of models, and the precision was assessed via bootstrapping. The achieved precisions are significantly better than the uncertainty of the dye position with respect to the macromolecule. The structure of the 4WJ was compared with FRET restrained structures of related RNA 3WJs, where one stem was removed. The types of 3WJs were studied: (I) without bulges, (II) with a small bulge (two unpaired nucleotides) and (III) with larger bulge (5 unpaired nucleotides). In conclusion the overall geometry of the RNA helices depends drastically on the junction type.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)[3] Kalinin, S. et al. Nat. Methods in press

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing the biomolecular structure. We apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements (eTCSPC) to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) which are derived from the hairpin ribozyme.Overall 283 FRET pairs were measured with single-molecule MFD and analyzed with the analysis toolkit [1] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.In order to study the influence of the junction on the RNA structure we studied the functional junction part with prolonged helices. Bulge and sequence variations were considered as dominant factors influencing junction conformations for RNA 3WJ. Six different RNA 3WJ with different sequences were studied, two of which have two and five unpaired nucleotides in the junction region.We found three different conformers for RNA 4WJ. However RNAs 3WJ have only one predominant conformer. Furthermore we report that bulges in the junction region determine orientation and rotation of helices, inducing coaxial stacking. Noteworthy the stacked helices are different for the 3WJs with different bulges. Our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality.[1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012).

RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of such stacking in case with no bulge. noteworthy the stacked helices are different for the 3WJs with two and five nucleotides in the bulge.Our studies showed that high precision FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers.References1. Sindbert S, et al. (2011) Accur ate distance determination of nucleic acids via Forster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133(8):2463-2480.2. Kalinin S,et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods in revision.