[{"publication":"iScience","has_accepted_license":"1","month":"03","article_number":"102139","oa_version":"Published Version","acknowledged_ssus":[{"_id":"EM-Fac"}],"project":[{"_id":"2564DBCA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"International IST Doctoral Program","grant_number":"665385"}],"language":[{"iso":"eng"}],"date_published":"2021-03-19T00:00:00Z","type":"journal_article","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode","short":"CC BY-NC-ND (4.0)","name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","image":"/images/cc_by_nc_nd.png"},"oa":1,"publication_identifier":{"eissn":["25890042"]},"status":"public","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","file":[{"checksum":"50585447386fe5842f07ab9b3a66e7e9","file_size":7431411,"date_created":"2021-03-03T07:38:14Z","content_type":"application/pdf","file_name":"2021_iScience_Kampjut.pdf","date_updated":"2021-03-03T07:38:14Z","access_level":"open_access","relation":"main_file","success":1,"creator":"dernst","file_id":"9219"}],"author":[{"first_name":"Domen","last_name":"Kampjut","full_name":"Kampjut, Domen","id":"37233050-F248-11E8-B48F-1D18A9856A87"},{"id":"3BB67EB0-F248-11E8-B48F-1D18A9856A87","first_name":"Julia","last_name":"Steiner","full_name":"Steiner, Julia"},{"id":"338D39FE-F248-11E8-B48F-1D18A9856A87","full_name":"Sazanov, Leonid A","orcid":"0000-0002-0977-7989","last_name":"Sazanov","first_name":"Leonid A"}],"issue":"3","pmid":1,"_id":"9205","scopus_import":"1","title":"Cryo-EM grid optimization for membrane proteins","intvolume":"        24","publication_status":"published","department":[{"_id":"LeSa"}],"article_processing_charge":"No","date_created":"2021-02-28T23:01:24Z","file_date_updated":"2021-03-03T07:38:14Z","quality_controlled":"1","ec_funded":1,"article_type":"original","publisher":"Elsevier","isi":1,"external_id":{"pmid":["33665558"],"isi":["000631646000012"]},"date_updated":"2023-08-07T13:54:06Z","citation":{"ista":"Kampjut D, Steiner J, Sazanov LA. 2021. Cryo-EM grid optimization for membrane proteins. iScience. 24(3), 102139.","mla":"Kampjut, Domen, et al. “Cryo-EM Grid Optimization for Membrane Proteins.” <i>IScience</i>, vol. 24, no. 3, 102139, Elsevier, 2021, doi:<a href=\"https://doi.org/10.1016/j.isci.2021.102139\">10.1016/j.isci.2021.102139</a>.","short":"D. Kampjut, J. Steiner, L.A. Sazanov, IScience 24 (2021).","chicago":"Kampjut, Domen, Julia Steiner, and Leonid A Sazanov. “Cryo-EM Grid Optimization for Membrane Proteins.” <i>IScience</i>. Elsevier, 2021. <a href=\"https://doi.org/10.1016/j.isci.2021.102139\">https://doi.org/10.1016/j.isci.2021.102139</a>.","ieee":"D. Kampjut, J. Steiner, and L. A. Sazanov, “Cryo-EM grid optimization for membrane proteins,” <i>iScience</i>, vol. 24, no. 3. Elsevier, 2021.","apa":"Kampjut, D., Steiner, J., &#38; Sazanov, L. A. (2021). Cryo-EM grid optimization for membrane proteins. <i>IScience</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.isci.2021.102139\">https://doi.org/10.1016/j.isci.2021.102139</a>","ama":"Kampjut D, Steiner J, Sazanov LA. Cryo-EM grid optimization for membrane proteins. <i>iScience</i>. 2021;24(3). doi:<a href=\"https://doi.org/10.1016/j.isci.2021.102139\">10.1016/j.isci.2021.102139</a>"},"year":"2021","abstract":[{"lang":"eng","text":"Cryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins."}],"doi":"10.1016/j.isci.2021.102139","day":"19","ddc":["570"],"volume":24,"acknowledgement":"We thank the Electron Microscopy Facilities at the Institute of Science and Technology Austria and at the Vienna Biocenter for providing access and training for the electron microscopes. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 665385 ."},{"publication":"eLife","has_accepted_license":"1","month":"07","article_number":"e59407","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"}],"oa_version":"Published Version","project":[{"_id":"26169496-B435-11E9-9278-68D0E5697425","name":"Revealing the functional mechanism of Mrp antiporter, an ancestor of complex I","grant_number":"24741"}],"language":[{"iso":"eng"}],"date_published":"2020-07-31T00:00:00Z","type":"journal_article","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","short":"CC BY (4.0)","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"oa":1,"publication_identifier":{"eissn":["2050084X"]},"status":"public","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","related_material":{"link":[{"url":"https://ist.ac.at/en/news/mystery-of-giant-proton-pump-solved/","description":"News on IST Homepage","relation":"press_release"}],"record":[{"status":"public","id":"8353","relation":"dissertation_contains"}]},"file":[{"file_name":"2020_eLife_Steiner.pdf","content_type":"application/pdf","date_updated":"2020-08-24T13:31:53Z","checksum":"b3656d14d5ddbb9d26e3074eea2d0c15","file_size":7320493,"date_created":"2020-08-24T13:31:53Z","creator":"cziletti","file_id":"8289","success":1,"access_level":"open_access","relation":"main_file"}],"author":[{"first_name":"Julia","last_name":"Steiner","orcid":"0000-0003-0493-3775","full_name":"Steiner, Julia","id":"3BB67EB0-F248-11E8-B48F-1D18A9856A87"},{"id":"338D39FE-F248-11E8-B48F-1D18A9856A87","first_name":"Leonid A","last_name":"Sazanov","orcid":"0000-0002-0977-7989","full_name":"Sazanov, Leonid A"}],"_id":"8284","pmid":1,"scopus_import":"1","title":"Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter","intvolume":"         9","publication_status":"published","department":[{"_id":"LeSa"}],"date_created":"2020-08-24T06:24:04Z","article_processing_charge":"No","file_date_updated":"2020-08-24T13:31:53Z","quality_controlled":"1","article_type":"original","publisher":"eLife Sciences Publications","isi":1,"external_id":{"isi":["000562123600001"],"pmid":["32735215"]},"date_updated":"2023-09-07T13:14:08Z","year":"2020","citation":{"ama":"Steiner J, Sazanov LA. Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter. <i>eLife</i>. 2020;9. doi:<a href=\"https://doi.org/10.7554/eLife.59407\">10.7554/eLife.59407</a>","apa":"Steiner, J., &#38; Sazanov, L. A. (2020). Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter. <i>ELife</i>. eLife Sciences Publications. <a href=\"https://doi.org/10.7554/eLife.59407\">https://doi.org/10.7554/eLife.59407</a>","ieee":"J. Steiner and L. A. Sazanov, “Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter,” <i>eLife</i>, vol. 9. eLife Sciences Publications, 2020.","chicago":"Steiner, Julia, and Leonid A Sazanov. “Structure and Mechanism of the Mrp Complex, an Ancient Cation/Proton Antiporter.” <i>ELife</i>. eLife Sciences Publications, 2020. <a href=\"https://doi.org/10.7554/eLife.59407\">https://doi.org/10.7554/eLife.59407</a>.","mla":"Steiner, Julia, and Leonid A. Sazanov. “Structure and Mechanism of the Mrp Complex, an Ancient Cation/Proton Antiporter.” <i>ELife</i>, vol. 9, e59407, eLife Sciences Publications, 2020, doi:<a href=\"https://doi.org/10.7554/eLife.59407\">10.7554/eLife.59407</a>.","short":"J. Steiner, L.A. Sazanov, ELife 9 (2020).","ista":"Steiner J, Sazanov LA. 2020. Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter. eLife. 9, e59407."},"abstract":[{"text":"Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.","lang":"eng"}],"doi":"10.7554/eLife.59407","day":"31","ddc":["570"],"acknowledgement":"This research was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Electron Microscopy Facility (EMF), the Life Science Facility (LSF) and the IST high-performance computing cluster. We thank Dr Victor-Valentin Hodirnau and Daniel Johann Gütl from IST Austria for assistance with collecting cryo-EM data. We thank Prof. Masahiro Ito (Graduate School of Life Sciences, Toyo University, Japan) for a kind provision of plasmid DNA encoding Mrp from A. flavithermus WK1. JS is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Science and Technology, Austria.","volume":9},{"doi":"10.15479/AT:ISTA:8353","degree_awarded":"PhD","day":"09","abstract":[{"lang":"eng","text":"Mrp (Multi resistance and pH adaptation) are broadly distributed secondary active antiporters that catalyze the transport of monovalent ions such as sodium and potassium outside of the cell coupled to the inward translocation of protons. Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG) encoded in a single operon, whereas other antiporters catalyzing the same reaction are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline environments and for reduction of the intracellular concentration of toxic cations. Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp subunits have primary sequence similarity to essential redox-driven proton pumps, such as respiratory complex I and membrane-bound hydrogenases. This similarity reinforces the hypothesis that these present day redox-driven proton pumps are descended from the Mrp antiporter. The Mrp structure serves as a model to understand the yet obscure coupling mechanism between ion or electron transfer and proton translocation in this large group of proteins. In the thesis, I am presenting the purification, biochemical analysis, cryo-EM analysis and molecular structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. Numerous conditions were screened to purify Mrp to high homogeneity and to obtain an appropriate distribution of single particles on cryo-EM grids covered with a continuous layer of ultrathin carbon. A preferred particle orientation problem was solved by performing a tilted data collection. The activity assays showed the specific pH-dependent\r\nprofile of secondary active antiporters. The molecular structure shows that Mrp is a dimer of seven-subunit protomers with 50 trans-membrane helices each. The dimer interface is built by many short and tilted transmembrane helices, probably causing a thinning of the bacterial membrane. The surface charge distribution shows an extraordinary asymmetry within each monomer, revealing presumable proton and sodium translocation pathways. The two largest\r\nand homologous Mrp subunits MrpA and MrpD probably translocate one proton each into the cell. The sodium ion is likely being translocated in the opposite direction within the small subunits along a ladder of charged and conserved residues. Based on the structure, we propose a mechanism were the antiport activity is accomplished via electrostatic interactions between the charged cations and key charged residues. The flexible key TM helices coordinate these\r\nelectrostatic interactions, while the membrane thinning between the monomers enables the translocation of sodium across the charged membrane. The entire family of redox-driven proton pumps is likely to perform their mechanism in a likewise manner."}],"date_updated":"2023-09-07T13:14:09Z","citation":{"ista":"Steiner J. 2020. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. Institute of Science and Technology Austria.","short":"J. Steiner, Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I, Institute of Science and Technology Austria, 2020.","mla":"Steiner, Julia. <i>Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I</i>. Institute of Science and Technology Austria, 2020, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:8353\">10.15479/AT:ISTA:8353</a>.","chicago":"Steiner, Julia. “Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I.” Institute of Science and Technology Austria, 2020. <a href=\"https://doi.org/10.15479/AT:ISTA:8353\">https://doi.org/10.15479/AT:ISTA:8353</a>.","ieee":"J. Steiner, “Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I,” Institute of Science and Technology Austria, 2020.","ama":"Steiner J. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. 2020. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:8353\">10.15479/AT:ISTA:8353</a>","apa":"Steiner, J. (2020). <i>Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:8353\">https://doi.org/10.15479/AT:ISTA:8353</a>"},"year":"2020","acknowledgement":"I acknowledge the scientific service units of the IST Austria for providing resources by the Life Science Facility, the Electron Microscopy Facility and the high-performance computer cluster. Special thanks to the cryo-EM specialists Valentin Hodirnau and Daniel Johann Gütl for spending many hours with me in front of the microscope and for supporting me to collect the data presented here. I also want to thank Professor Masahiro Ito for providing plasmid DNA\r\nencoding Mrp from Anoxybacillus flavithermus WK1. I am a recipient of a DOC Fellowship of the Austrian Academy of Sciences.","ddc":["572"],"publication_status":"published","article_processing_charge":"No","department":[{"_id":"LeSa"}],"date_created":"2020-09-09T14:27:01Z","alternative_title":["ISTA Thesis"],"title":"Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I","_id":"8353","author":[{"last_name":"Steiner","first_name":"Julia","full_name":"Steiner, Julia","orcid":"0000-0003-0493-3775","id":"3BB67EB0-F248-11E8-B48F-1D18A9856A87"}],"publisher":"Institute of Science and Technology Austria","page":"191","file_date_updated":"2021-09-16T12:40:56Z","publication_identifier":{"issn":["2663-337X"]},"supervisor":[{"orcid":"0000-0002-0977-7989","full_name":"Sazanov, Leonid A","first_name":"Leonid A","last_name":"Sazanov","id":"338D39FE-F248-11E8-B48F-1D18A9856A87"}],"oa":1,"date_published":"2020-09-09T00:00:00Z","type":"dissertation","file":[{"relation":"main_file","access_level":"open_access","file_id":"8354","creator":"jsteiner","date_created":"2020-09-09T14:22:35Z","file_size":117547589,"checksum":"2388d7e6e7a4d364c096fa89f305c3de","date_updated":"2021-09-16T12:40:56Z","file_name":"Thesis_Julia_Steiner_pdfA.pdf","content_type":"application/pdf"},{"file_name":"Thesis_Julia_Steiner.docx","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","date_updated":"2020-09-15T08:48:37Z","file_size":223328668,"checksum":"ba112f957b7145462d0ab79044873ee9","date_created":"2020-09-09T14:23:25Z","creator":"jsteiner","file_id":"8355","access_level":"closed","relation":"source_file"}],"related_material":{"record":[{"relation":"part_of_dissertation","id":"8284","status":"public"}]},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","status":"public","oa_version":"None","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"}],"project":[{"_id":"26169496-B435-11E9-9278-68D0E5697425","name":"Revealing the functional mechanism of Mrp antiporter, an ancestor of complex I","grant_number":"24741"}],"month":"09","has_accepted_license":"1","language":[{"iso":"eng"}]}]