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      A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

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          Abstract

          Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample’s ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

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          An Algorithm for Least-Squares Estimation of Nonlinear Parameters

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            A method for the solution of certain non-linear problems in least squares

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              Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.

              Much of life's essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draft
                Role: ConceptualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2021
                20 January 2021
                : 16
                : 1
                : e0245693
                Affiliations
                [1 ] Institute of Applied Physics, TU Wien, Vienna, Austria
                [2 ] Faculty of Mathematics, University of Vienna, Vienna, Austria
                Texas A&M University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-3771-8353
                https://orcid.org/0000-0002-3622-7077
                https://orcid.org/0000-0001-9527-2970
                https://orcid.org/0000-0001-9378-7452
                https://orcid.org/0000-0003-1542-1089
                Article
                PONE-D-20-25964
                10.1371/journal.pone.0245693
                7817001
                33471861
                60cb303b-133c-4af7-866d-e7b0dfbd5f97
                © 2021 Schneider 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.

                History
                : 19 August 2020
                : 5 January 2021
                Page count
                Figures: 7, Tables: 0, Pages: 23
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100002428, Austrian Science Fund;
                Award ID: F6807-N36
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002428, Austrian Science Fund;
                Award ID: F6809-N36
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002428, Austrian Science Fund;
                Award ID: I3661-N27
                Award Recipient :
                OS: F6807-N36, FWF, https://www.fwf.ac.at/ OS: I3661-N27, FWF, https://www.fwf.ac.at/ GJS: F6809-N36, FWF, https://www.fwf.ac.at/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Physical Sciences
                Materials Science
                Materials
                Oligomers
                Physical Sciences
                Physics
                Particle Physics
                Elementary Particles
                Photons
                Computer and Information Sciences
                Information Theory
                Background Signal Noise
                Engineering and Technology
                Signal Processing
                Background Signal Noise
                Physical Sciences
                Mathematics
                Geometry
                Radii
                Physical Sciences
                Mathematics
                Probability Theory
                Random Variables
                Physical Sciences
                Physics
                Electromagnetic Radiation
                Light
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Signaling Molecules
                Physical Sciences
                Physics
                Optics
                Focal Planes
                Custom metadata
                All relevant data are within the manuscript and its Supporting information files.

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