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The practical application of new single molecule protein sequencing (SMPS) technologies requires accurate estimates of their associated sequencing error rates. Here, we describe the development and application of two distinct parameter estimation methods for analyzing SMPS reads produced by fluorosequencing. A Hidden Markov Model (HMM) based approach, extends whatprot, where we previously used HMMs for SMPS peptide-read matching. This extension offers a principled approach for estimating key parameters for fluorosequencing experiments, including missed amino acid cleavages, dye loss, and peptide detachment. Specifically, we adapted the Baum-Welch algorithm, a standard technique to estimate transition probabilities for an HMM using expectation maximization, but modified here to estimate a small number of parameter values directly rather than estimating every transition probability independently. We demonstrate a high degree of accuracy on simulated data, but on experimental datasets, we observed that the model needed to be augmented with an additional error type, N-terminal blocking. This, in combination with data pre-processing, results in reasonable parameterizations of experimental datasets that agree with controlled experimental perturbations. A second independent implementation using a hybrid of DIRECT and Powell's method to reduce the root mean squared error (RMSE) between simulations and the real dataset was also developed. We compare these methods on both simulated and real data, finding that our Baum-Welch based approach outperforms DIRECT and Powell's method by most, but not all, criteria. Although some discrepancies between the results exist, we also find that both approaches provide similar error rate estimates from experimental single molecule fluorosequencing datasets., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.M.B. and E.M.M. are co-founders and shareholders of Erisyon, Inc., and are co-inventors on granted patents or pending patent applications related to single-molecule protein sequencing. A.M.B. serves on the board of directors and E.M.M. serves on the scientific advisory board. M.B.S., K.V., T.B., H.D.S., J.H.M., T.M.F., C.M., and A.M.B. are affiliated with Erisyon, Inc., as employees or shareholders. H.D.S. is currently employed by UT Austin with funding from a Sponsored Research Agreement from Erisyon, Inc., (Copyright: © 2024 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice ( Oryza sativa ; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation., Competing Interests: The authors declare no conflict of interest.