Rachael Kretsch

All Publications

• RNA target highlights in CASP15: Evaluation of predicted models by structure providers. Proteins Kretsch, R. C., Andersen, E. S., Bujnicki, J. M., Chiu, W., Das, R., Luo, B., Masquida, B., McRae, E. K., Schroeder, G. M., Su, Z., Wedekind, J. E., Xu, L., Zhang, K., Zheludev, I. N., Moult, J., Kryshtafovych, A. 2023

  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.

  View details for DOI 10.1002/prot.26550

  View details for PubMedID 37466021

• New prediction categories in CASP15. Proteins Kryshtafovych, A., Antczak, M., Szachniuk, M., Zok, T., Kretsch, R. C., Rangan, R., Pham, P., Das, R., Robin, X., Studer, G., Durairaj, J., Eberhardt, J., Sweeney, A., Topf, M., Schwede, T., Fidelis, K., Moult, J. 2023

  Abstract

  Prediction categories in the Critical Assessment of Structure Prediction (CASP) experiments change with the need to address specific problems in structure modeling. In CASP15, four new prediction categories were introduced: RNA structure, ligand-protein complexes, accuracy of oligomeric structures and their interfaces, and ensembles of alternative conformations. This paper lists technical specifications for these categories and describes their integration in the CASP data management system.

  View details for DOI 10.1002/prot.26515

  View details for PubMedID 37306011

• Assessment of three-dimensional RNA structure prediction in CASP15. bioRxiv : the preprint server for biology Das, R., Kretsch, R. C., Simpkin, A., Mulvaney, T., Pham, P., Rangan, R., Bu, F., Keegan, R., Topf, M., Rigden, D., Miao, Z., Westhof, E. 2023

  Abstract

  The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report double-blind assessments of RNA structure predictions in CASP15, the first CASP exercise in which RNA modeling was assessed. Forty two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, who did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and X-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global topology of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as non-canonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.

  View details for DOI 10.1101/2023.04.25.538330

  View details for PubMedID 37162955

• Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nature structural & molecular biology Zhang, K., Zheludev, I. N., Hagey, R. J., Haslecker, R., Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R., Kladwang, W., Li, S., Wu, M. T., Pham, E. A., Bernardin-Souibgui, C., Baric, R. S., Sheahan, T. P., D'Souza, V., Glenn, J. S., Chiu, W., Das, R. 2021

  Abstract

  Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9A resolution cryo-EM structure of the FSE (88nucleotides, ~28kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100nM concentration.

  View details for DOI 10.1038/s41594-021-00653-y

  View details for PubMedID 34426697

• Interpretation of RNA cryo-EM maps of various resolutions Kretsch, R., Das, R., Chiu, W. INT UNION CRYSTALLOGRAPHY. 2021: A217

  View details for DOI 10.1107/S0108767321097828

  View details for Web of Science ID 000720840500218

• De novo 3D models of SARS-CoV-2 RNA elements from consensus experimental secondary structures. Nucleic acids research Rangan, R., Watkins, A. M., Chacon, J., Kretsch, R., Kladwang, W., Zheludev, I. N., Townley, J., Rynge, M., Thain, G., Das, R. 2021

  Abstract

  The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta's FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5' UTR; the reverse complement of the 5' UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3' UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m and 3' UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ('FARFAR2-SARS-CoV-2', https://github.com/DasLab/FARFAR2-SARS-CoV-2; and 'FARFAR2-Apo-Riboswitch', at https://github.com/DasLab/FARFAR2-Apo-Riboswitch') include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.

  View details for DOI 10.1093/nar/gkab119

  View details for PubMedID 33693814

• Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms. Nature Billesbølle, C. B., Azumaya, C. M., Kretsch, R. C., Powers, A. S., Gonen, S. n., Schneider, S. n., Arvedson, T. n., Dror, R. O., Cheng, Y. n., Manglik, A. n. 2020

  Abstract

  The serum iron level in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing ferroportin internalization and degradation1. Aberrant ferroportin activity can lead to diseases of iron overload, such as hemochromatosis, or iron limitation anemias2. Here, we determined cryogenic electron microscopy (cryo-EM) structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with cobalt, an iron mimetic, and hepcidin. These structures and accompanying molecular dynamics simulations identify two metal binding sites within the N- and C-domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy-terminus of hepcidin directly contacts the divalent metal in the ferroportin C-domain. We further show that hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, where only iron loaded ferroportin molecules are targeted for degradation. More broadly, our structural and functional insights are likely to enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

  View details for DOI 10.1038/s41586-020-2668-z

  View details for PubMedID 32814342