DOI: 10.7554/elife.42747. Protein denaturation at the air-water interface and how to prevent it

Protein denaturation at the air-water interface and how to prevent it

10.7554/elife.42747

Crossref journal-article

eLife Sciences Publications, Ltd

eLife (4374)

Abstract

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Bibliography

DâImprima, E., Floris, D., Joppe, M., SÃ¡nchez, R., Grininger, M., & KÃ¼hlbrandt, W. (2019). Protein denaturation at the air-water interface and how to prevent it. ELife, 8. CLOCKSS.

Authors 6

Edoardo D'Imprima (first)
Davide Floris (additional)
Mirko Joppe (additional)
Ricardo Sánchez (additional)
Martin Grininger (additional)
Werner Kühlbrandt (additional)

References 72 Referenced 226

10.7554/eLife.00461 / eLife / Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles by Bai (2013)
10.1016/j.tibs.2014.10.005 / Trends in Biochemical Sciences / How cryo-EM is revolutionizing structural biology by Bai (2015)
10.1038/nature14892 / Nature / An atomic structure of human γ-secretase by Bai (2015)
10.1038/nature24627 / Nature / Structure of the human MHC-I peptide-loading complex by Blees (2017)
10.1016/j.jmb.2012.12.021 / Journal of Molecular Biology / 7.5-Å cryo-em structure of the mycobacterial fatty acid synthase by Boehringer (2013)
10.1038/nsmb.3386 / Nature Structural & Molecular Biology / Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution by Boland (2017)
10.1016/S0304-3991(97)00126-5 / Ultramicroscopy / Evaluation of charging on macromolecules in electron cryomicroscopy by Brink (1998)
10.1107/S2059798317003369 / Acta Crystallographica Section D Structural Biology / The dynamo package for tomography and subtomogram averaging: components for MATLAB, GPU computing and EC2 amazon web services by Castaño-Díez (2017)
10.1073/pnas.0406901101 / PNAS / Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain by Chakravarty (2004)
10.1016/j.ultramic.2013.06.004 / Ultramicroscopy / High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy by Chen (2013)
10.1038/nmeth.4405 / Nature Methods / Convolutional neural networks for automated annotation of cellular cryo-electron tomograms by Chen (2017)
10.1016/j.cell.2015.03.050 / Cell / A primer to single-particle cryo-electron microscopy by Cheng (2015)
10.7554/eLife.30483 / eLife / Cryo-EM structure of the bifunctional secretin complex of thermus thermophilus by D'Imprima (2017)
10.1016/j.jsb.2018.01.002 / Journal of Structural Biology / Spotiton: new features and applications by Dandey (2018)
10.1017/S0033583500004297 / Quarterly Reviews of Biophysics / Cryo-electron microscopy of vitrified specimens by Dubochet (1988)
10.1038/nature13916 / Nature / Architecture and conformational switch mechanism of the ryanodine receptor by Efremov (2015)
10.1046/j.1432-1327.2000.01282.x / European Journal of Biochemistry / A novel function of yeast fatty acid synthase. subunit alpha is capable of self-pantetheinylation by Fichtlscherer (2000)
10.1006/jsbi.2001.4406 / Journal of Structural Biology / Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion by Frangakis (2001)
10.1038/nchembio.2314 / Nature Chemical Biology / Engineering fatty acid synthases for directed polyketide production by Gajewski (2017)
10.1038/ncomms14650 / Nature Communications / Engineering fungal de novo fatty acid synthesis for short chain fatty acid production by Gajewski (2017)
10.1038/nmat1849 / Nature Materials / The rise of graphene by Geim (2007)
10.1073/pnas.0913547107 / PNAS / Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy by Gipson (2010)
10.1016/j.cocis.2017.12.009 / Current Opinion in Colloid & Interface Science / Proteins, interfaces, and cryo-em grids by Glaeser (2018)
10.1007/s41048-016-0026-3 / Biophysics Reports / Opinion: hazards faced by macromolecules when confined to thin aqueous films by Glaeser (2017)
10.1002/pro.3235 / Protein Science / UCSF ChimeraX: meeting modern challenges in visualization and analysis by Goddard (2018)
10.7554/eLife.06980 / eLife / Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6 by Grant (2015)
10.1016/j.jsb.2016.06.007 / Journal of Structural Biology / Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging by Hagen (2017)
10.1016/j.jsb.2017.02.009 / Journal of Structural Biology / Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality by Han (2017)
{'volume-title': 'Intermolecular and Surface Forces', 'year': '2011', 'author': 'Israelachvili', 'key': 'bib29'} / Intermolecular and Surface Forces by Israelachvili (2011)
10.1016/j.jsb.2012.04.020 / Journal of Structural Biology / Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM by Jain (2012)
10.1126/science.1138248 / Science / Structure of fungal fatty acid synthase and implications for iterative substrate shuttling by Jenni (2007)
10.1073/pnas.0805827105 / PNAS / Inhibition of the fungal fatty acid synthase type I multienzyme complex by Johansson (2008)
10.15252/msb.20167412 / Molecular Systems Biology / Capturing protein communities by structural proteomics in a thermophilic eukaryote by Kastritis (2017)
10.1126/science.aar7389 / Science / Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation by Kim (2018)
10.7554/eLife.18722 / eLife / Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2 by Kimanius (2016)
10.1006/jsbi.1996.0013 / Journal of Structural Biology / Computer visualization of three-dimensional image data using IMOD by Kremer (1996)
10.1126/science.1251652 / Science / The resolution revolution by Kuhlbrandt (2014)
10.1016/j.jsb.2011.02.005 / Journal of Structural Biology / The surface of evaporated carbon films is an insulating, high-bandgap material by Larson (2011)
10.1016/j.cell.2007.03.013 / Cell / The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together by Lomakin (2007)
10.1016/j.jsb.2005.07.007 / Journal of Structural Biology / Automated electron microscope tomography using robust prediction of specimen movements by Mastronarde (2005)
10.1111/j.1365-2818.1983.tb04225.x / Journal of Microscopy / Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples by McDowall (1983)
10.1038/s41467-017-00782-3 / Nature Communications / Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy by Naydenova (2017)
10.1021/cr60076a001 / Chemical Reviews / The Surface Activity of Proteins by Neurath (1938)
10.1038/nature14548 / Nature / The architecture of the spliceosomal U4/U6.U5 tri-snRNP by Nguyen (2015)
10.7554/eLife.34257 / eLife / Routine single particle CryoEM sample and grid characterization by tomography by Noble (2018)
10.1073/pnas.63.4.1377 / PNAS / Crystallization of a multienzyme complex: fatty acid synthetase from yeast by Oesterhelt (1969)
10.1016/j.jsb.2009.12.020 / Journal of Structural Biology / Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples by Pantelic (2010)
10.1016/j.ssc.2012.04.038 / Solid State Communications / The application of graphene as a sample support in transmission electron microscopy by Pantelic (2012)
10.1063/1.4870531 / Applied Physics Letters / Rendering graphene supports hydrophilic with non-covalent aromatic functionalization for transmission electron microscopy by Pantelic (2014)
10.1016/bs.mie.2016.04.011 / Methods in Enzymology / Specimen preparation for High-Resolution Cryo-EM by Passmore (2016)
10.1002/jcc.20084 / Journal of Computational Chemistry / UCSF chimera--a visualization system for exploratory research and analysis by Pettersen (2004)
10.1146/annurev.biochem.052208.114057 / Annual Review of Biochemistry / Amphipols, Nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions by Popot (2010)
10.1017/S0033583500002900 / Quarterly Reviews of Biophysics / Experimental methods for investigating protein adsorption kinetics at surfaces by Ramsden (1994)
10.1016/j.jsb.2015.08.008 / Journal of Structural Biology / CTFFIND4: fast and accurate defocus estimation from electron micrographs by Rohou (2015)
10.1038/nmeth.2931 / Nature Methods / Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas by Russo (2014)
10.1016/j.jsb.2013.04.014 / Journal of Structural Biology / Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging by Sader (2013)
10.1074/jbc.M116.724153 / Journal of Biological Chemistry / Topology and structure/Function correlation of ring- and Gate-forming domains in the dynamic secretin complex of thermus thermophilus by Salzer (2016)
10.1038/nature26153 / Nature / Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis by Scapin (2018)
10.1038/nmeth.2115 / Nature Methods / Prevention of overfitting in cryo-EM structure determination by Scheres (2012)
10.1126/science.1199358 / Science / Three-dimensional model of salmonella's needle complex at subnanometer resolution by Schraidt (2011)
10.1098/rsob.170255 / Open Biology / Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence by Takizawa (2018)
10.1016/j.jsb.2006.05.009 / Journal of Structural Biology / EMAN2: an extensible image processing suite for electron microscopy by Tang (2007)
10.1016/j.jsb.2008.06.004 / Journal of Structural Biology / Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future by Taylor (2008)
10.1016/0095-8522(60)90002-7 / Journal of Colloid Science / A theory and method for the spreading of protein monolayers by Trurnit (1960)
10.1016/j.jsb.2018.01.001 / Journal of Structural Biology / Optimizing "self-wicking" nanowire grids by Wei (2018)
10.1111/j.1432-1033.1979.tb12885.x / European Journal of Biochemistry / Studies on the multi-enzyme complex of yeast fatty-acid synthetase. reversible dissociation and isolation of two polypeptide chains by Wieland (1979)
10.1038/s41591-018-0042-6 / Nature Medicine / Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1 by Xu (2018)
10.1016/j.cell.2017.06.039 / Cell / Structure of the Nav1.4-β1 complex from electric eel by Yan (2017)
10.1021/la00021a062 / Langmuir / Two-Dimensional protein array growth in thin layers of protein solution on aqueous subphases by Yoshimura (1994)
10.1016/j.jsb.2015.11.003 / Journal of Structural Biology / Gctf: real-time CTF determination and correction by Zhang (2016)
10.1038/nmeth.4193 / Nature Methods / MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy by Zheng (2017)
10.1038/s41586-018-0255-3 / Nature / Structure of a human synaptic GABAA receptor by Zhu (2018)

Dates

Type	When
Created	6 years, 5 months ago (April 1, 2019, 7 a.m.)
Deposited	1 year, 10 months ago (Oct. 11, 2023, 8:52 p.m.)
Indexed	45 minutes ago (Sept. 2, 2025, 8:01 p.m.)
Issued	6 years, 5 months ago (April 1, 2019)
Published	6 years, 5 months ago (April 1, 2019)
Published Online	6 years, 5 months ago (April 1, 2019)

Funders 6

Max-Planck-Gesellschaft 10.13039/501100004189
Region: Europe
gov (Research institutes and centers)
Labels4
1. Max Planck Society for the Advancement of Science
2. Max-Planck-Gesellschaft zur Förderung der Wissenschaften
3. Max Planck Society
4. MPG
Max-Planck-Gesellschaft 10.13039/501100004189
Region: Europe
gov (Research institutes and centers)
Labels4
1. Max Planck Society for the Advancement of Science
2. Max-Planck-Gesellschaft zur Förderung der Wissenschaften
3. Max Planck Society
4. MPG
Volkswagen Foundation 10.13039/501100001663
Region: Europe
pri (Trusts, charities, foundations (both public and private))
Labels1
1. VolkswagenStiftung
Alexander von Humboldt Foundation 10.13039/100005156 Alexander von Humboldt-Stiftung
Region: Europe
pri (Trusts, charities, foundations (both public and private))
Labels5
1. Humboldt-Stiftung
2. Humboldt Foundation
3. Alexander von Humboldt Foundation
4. Humboldt Stiftung
5. AvH
Awards1
1. Sofja Kovalevskaja Award to Mikhail Kudryashev
Deutsche Forschungsgemeinschaft 10.13039/501100001659
Region: Europe
gov (National government)
Labels3
1. German Research Association
2. German Research Foundation
3. DFG
Awards1
1. SFB807
Volkswagen Foundation 10.13039/501100001663
Region: Europe
pri (Trusts, charities, foundations (both public and private))
Labels1
1. VolkswagenStiftung
Awards1
1. 85701

BibTeX

@article{D_Imprima_2019, title={Protein denaturation at the air-water interface and how to prevent it}, volume={8}, ISSN={2050-084X}, url={http://dx.doi.org/10.7554/elife.42747}, DOI={10.7554/elife.42747}, journal={eLife}, publisher={eLife Sciences Publications, Ltd}, author={D’Imprima, Edoardo and Floris, Davide and Joppe, Mirko and Sánchez, Ricardo and Grininger, Martin and Kühlbrandt, Werner}, year={2019}, month=apr }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        9,
        3
      ]
    ],
    "date-time": "2025-09-03T00:01:22Z",
    "timestamp": 1756857682230,
    "version": "3.41.2"
  },
  "reference-count": 72,
  "publisher": "eLife Sciences Publications, Ltd",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2019,
            4,
            1
          ]
        ],
        "date-time": "2019-04-01T00:00:00Z",
        "timestamp": 1554076800000
      },
      "content-version": "vor",
      "delay-in-days": 0,
      "URL": "http://creativecommons.org/licenses/by/4.0/"
    },
    {
      "start": {
        "date-parts": [
          [
            2019,
            4,
            1
          ]
        ],
        "date-time": "2019-04-01T00:00:00Z",
        "timestamp": 1554076800000
      },
      "content-version": "am",
      "delay-in-days": 0,
      "URL": "http://creativecommons.org/licenses/by/4.0/"
    },
    {
      "start": {
        "date-parts": [
          [
            2019,
            4,
            1
          ]
        ],
        "date-time": "2019-04-01T00:00:00Z",
        "timestamp": 1554076800000
      },
      "content-version": "tdm",
      "delay-in-days": 0,
      "URL": "http://creativecommons.org/licenses/by/4.0/"
    }
  ],
  "funder": [
    {
      "DOI": "10.13039/501100004189",
      "name": "Max-Planck-Gesellschaft",
      "doi-asserted-by": "publisher",
      "id": [
        {
          "id": "10.13039/501100004189",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    },
    {
      "DOI": "10.13039/501100004189",
      "name": "Max-Planck-Gesellschaft",
      "doi-asserted-by": "publisher",
      "id": [
        {
          "id": "10.13039/501100004189",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    },
    {
      "DOI": "10.13039/501100001663",
      "name": "Volkswagen Foundation",
      "doi-asserted-by": "publisher",
      "id": [
        {
          "id": "10.13039/501100001663",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    },
    {
      "DOI": "10.13039/100005156",
      "name": "Alexander von Humboldt Foundation",
      "doi-asserted-by": "publisher",
      "award": [
        "Sofja Kovalevskaja Award to Mikhail Kudryashev"
      ],
      "id": [
        {
          "id": "10.13039/100005156",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    },
    {
      "DOI": "10.13039/501100001659",
      "name": "Deutsche Forschungsgemeinschaft",
      "doi-asserted-by": "publisher",
      "award": [
        "SFB807"
      ],
      "id": [
        {
          "id": "10.13039/501100001659",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    },
    {
      "DOI": "10.13039/501100001663",
      "name": "Volkswagen Foundation",
      "doi-asserted-by": "publisher",
      "award": [
        "85701"
      ],
      "id": [
        {
          "id": "10.13039/501100001663",
          "id-type": "DOI",
          "asserted-by": "publisher"
        }
      ]
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "abstract": "<jats:p>Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.</jats:p>",
  "DOI": "10.7554/elife.42747",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2019,
        4,
        1
      ]
    ],
    "date-time": "2019-04-01T11:00:29Z",
    "timestamp": 1554116429000
  },
  "source": "Crossref",
  "is-referenced-by-count": 226,
  "title": "Protein denaturation at the air-water interface and how to prevent it",
  "prefix": "10.7554",
  "volume": "8",
  "author": [
    {
      "ORCID": "https://orcid.org/0000-0002-9830-7929",
      "authenticated-orcid": true,
      "given": "Edoardo",
      "family": "D'Imprima",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt, Germany"
        }
      ]
    },
    {
      "ORCID": "https://orcid.org/0000-0001-9144-5459",
      "authenticated-orcid": true,
      "given": "Davide",
      "family": "Floris",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt, Germany"
        }
      ]
    },
    {
      "ORCID": "https://orcid.org/0000-0001-6463-0253",
      "authenticated-orcid": true,
      "given": "Mirko",
      "family": "Joppe",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Buchmann Institute for Molecular Life Sciences, Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt, Germany"
        }
      ]
    },
    {
      "given": "Ricardo",
      "family": "S\u00e1nchez",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Sofja Kovalevskaja Group, Max Planck Institute of Biophysics, Frankfurt, Germany"
        }
      ]
    },
    {
      "given": "Martin",
      "family": "Grininger",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Buchmann Institute for Molecular Life Sciences, Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt, Germany"
        }
      ]
    },
    {
      "ORCID": "https://orcid.org/0000-0002-2013-4810",
      "authenticated-orcid": true,
      "given": "Werner",
      "family": "K\u00fchlbrandt",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt, Germany"
        }
      ]
    }
  ],
  "member": "4374",
  "published-online": {
    "date-parts": [
      [
        2019,
        4,
        1
      ]
    ]
  },
  "reference": [
    {
      "key": "bib1",
      "doi-asserted-by": "publisher",
      "DOI": "10.7554/eLife.00461",
      "article-title": "Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles",
      "volume": "2",
      "author": "Bai",
      "year": "2013",
      "journal-title": "eLife"
    },
    {
      "key": "bib2",
      "doi-asserted-by": "publisher",
      "first-page": "49",
      "DOI": "10.1016/j.tibs.2014.10.005",
      "article-title": "How cryo-EM is revolutionizing structural biology",
      "volume": "40",
      "author": "Bai",
      "year": "2015",
      "journal-title": "Trends in Biochemical Sciences"
    },
    {
      "key": "bib3",
      "doi-asserted-by": "publisher",
      "first-page": "212",
      "DOI": "10.1038/nature14892",
      "article-title": "An atomic structure of human \u03b3-secretase",
      "volume": "525",
      "author": "Bai",
      "year": "2015",
      "journal-title": "Nature"
    },
    {
      "key": "bib4",
      "doi-asserted-by": "publisher",
      "first-page": "525",
      "DOI": "10.1038/nature24627",
      "article-title": "Structure of the human MHC-I peptide-loading complex",
      "volume": "551",
      "author": "Blees",
      "year": "2017",
      "journal-title": "Nature"
    },
    {
      "key": "bib5",
      "doi-asserted-by": "publisher",
      "first-page": "841",
      "DOI": "10.1016/j.jmb.2012.12.021",
      "article-title": "7.5-\u00c5 cryo-em structure of the mycobacterial fatty acid synthase",
      "volume": "425",
      "author": "Boehringer",
      "year": "2013",
      "journal-title": "Journal of Molecular Biology"
    },
    {
      "key": "bib6",
      "doi-asserted-by": "publisher",
      "first-page": "414",
      "DOI": "10.1038/nsmb.3386",
      "article-title": "Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution",
      "volume": "24",
      "author": "Boland",
      "year": "2017",
      "journal-title": "Nature Structural & Molecular Biology"
    },
    {
      "key": "bib7",
      "doi-asserted-by": "publisher",
      "first-page": "41",
      "DOI": "10.1016/S0304-3991(97)00126-5",
      "article-title": "Evaluation of charging on macromolecules in electron cryomicroscopy",
      "volume": "72",
      "author": "Brink",
      "year": "1998",
      "journal-title": "Ultramicroscopy"
    },
    {
      "key": "bib8",
      "doi-asserted-by": "publisher",
      "first-page": "478",
      "DOI": "10.1107/S2059798317003369",
      "article-title": "The dynamo package for tomography and subtomogram averaging: components for MATLAB, GPU computing and EC2 amazon web services",
      "volume": "73",
      "author": "Casta\u00f1o-D\u00edez",
      "year": "2017",
      "journal-title": "Acta Crystallographica Section D Structural Biology"
    },
    {
      "key": "bib9",
      "doi-asserted-by": "publisher",
      "first-page": "15567",
      "DOI": "10.1073/pnas.0406901101",
      "article-title": "Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain",
      "volume": "101",
      "author": "Chakravarty",
      "year": "2004",
      "journal-title": "PNAS"
    },
    {
      "key": "bib10",
      "doi-asserted-by": "publisher",
      "first-page": "24",
      "DOI": "10.1016/j.ultramic.2013.06.004",
      "article-title": "High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy",
      "volume": "135",
      "author": "Chen",
      "year": "2013",
      "journal-title": "Ultramicroscopy"
    },
    {
      "key": "bib11",
      "doi-asserted-by": "publisher",
      "first-page": "983",
      "DOI": "10.1038/nmeth.4405",
      "article-title": "Convolutional neural networks for automated annotation of cellular cryo-electron tomograms",
      "volume": "14",
      "author": "Chen",
      "year": "2017",
      "journal-title": "Nature Methods"
    },
    {
      "key": "bib12",
      "doi-asserted-by": "publisher",
      "first-page": "438",
      "DOI": "10.1016/j.cell.2015.03.050",
      "article-title": "A primer to single-particle cryo-electron microscopy",
      "volume": "161",
      "author": "Cheng",
      "year": "2015",
      "journal-title": "Cell"
    },
    {
      "key": "bib13",
      "doi-asserted-by": "publisher",
      "DOI": "10.7554/eLife.30483",
      "article-title": "Cryo-EM structure of the bifunctional secretin complex of thermus thermophilus",
      "volume": "6",
      "author": "D'Imprima",
      "year": "2017",
      "journal-title": "eLife"
    },
    {
      "key": "bib14",
      "doi-asserted-by": "publisher",
      "first-page": "161",
      "DOI": "10.1016/j.jsb.2018.01.002",
      "article-title": "Spotiton: new features and applications",
      "volume": "202",
      "author": "Dandey",
      "year": "2018",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib15",
      "doi-asserted-by": "publisher",
      "first-page": "129",
      "DOI": "10.1017/S0033583500004297",
      "article-title": "Cryo-electron microscopy of vitrified specimens",
      "volume": "21",
      "author": "Dubochet",
      "year": "1988",
      "journal-title": "Quarterly Reviews of Biophysics"
    },
    {
      "key": "bib16",
      "doi-asserted-by": "publisher",
      "first-page": "39",
      "DOI": "10.1038/nature13916",
      "article-title": "Architecture and conformational switch mechanism of the ryanodine receptor",
      "volume": "517",
      "author": "Efremov",
      "year": "2015",
      "journal-title": "Nature"
    },
    {
      "key": "bib17",
      "doi-asserted-by": "publisher",
      "first-page": "2666",
      "DOI": "10.1046/j.1432-1327.2000.01282.x",
      "article-title": "A novel function of yeast fatty acid synthase. subunit alpha is capable of self-pantetheinylation",
      "volume": "267",
      "author": "Fichtlscherer",
      "year": "2000",
      "journal-title": "European Journal of Biochemistry"
    },
    {
      "key": "bib18",
      "doi-asserted-by": "publisher",
      "first-page": "239",
      "DOI": "10.1006/jsbi.2001.4406",
      "article-title": "Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion",
      "volume": "135",
      "author": "Frangakis",
      "year": "2001",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib19",
      "doi-asserted-by": "publisher",
      "first-page": "363",
      "DOI": "10.1038/nchembio.2314",
      "article-title": "Engineering fatty acid synthases for directed polyketide production",
      "volume": "13",
      "author": "Gajewski",
      "year": "2017",
      "journal-title": "Nature Chemical Biology"
    },
    {
      "key": "bib20",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/ncomms14650",
      "article-title": "Engineering fungal de novo fatty acid synthesis for short chain fatty acid production",
      "volume": "8",
      "author": "Gajewski",
      "year": "2017",
      "journal-title": "Nature Communications"
    },
    {
      "key": "bib21",
      "doi-asserted-by": "publisher",
      "first-page": "183",
      "DOI": "10.1038/nmat1849",
      "article-title": "The rise of graphene",
      "volume": "6",
      "author": "Geim",
      "year": "2007",
      "journal-title": "Nature Materials"
    },
    {
      "key": "bib22",
      "doi-asserted-by": "publisher",
      "first-page": "9164",
      "DOI": "10.1073/pnas.0913547107",
      "article-title": "Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy",
      "volume": "107",
      "author": "Gipson",
      "year": "2010",
      "journal-title": "PNAS"
    },
    {
      "key": "bib23",
      "doi-asserted-by": "publisher",
      "first-page": "1",
      "DOI": "10.1016/j.cocis.2017.12.009",
      "article-title": "Proteins, interfaces, and cryo-em grids",
      "volume": "34",
      "author": "Glaeser",
      "year": "2018",
      "journal-title": "Current Opinion in Colloid & Interface Science"
    },
    {
      "key": "bib24",
      "doi-asserted-by": "publisher",
      "first-page": "1",
      "DOI": "10.1007/s41048-016-0026-3",
      "article-title": "Opinion: hazards faced by macromolecules when confined to thin aqueous films",
      "volume": "3",
      "author": "Glaeser",
      "year": "2017",
      "journal-title": "Biophysics Reports"
    },
    {
      "key": "bib25",
      "doi-asserted-by": "publisher",
      "first-page": "14",
      "DOI": "10.1002/pro.3235",
      "article-title": "UCSF ChimeraX: meeting modern challenges in visualization and analysis",
      "volume": "27",
      "author": "Goddard",
      "year": "2018",
      "journal-title": "Protein Science"
    },
    {
      "key": "bib26",
      "doi-asserted-by": "publisher",
      "DOI": "10.7554/eLife.06980",
      "article-title": "Measuring the optimal exposure for single particle cryo-EM using a 2.6 \u00c5 reconstruction of rotavirus VP6",
      "volume": "4",
      "author": "Grant",
      "year": "2015",
      "journal-title": "eLife"
    },
    {
      "key": "bib27",
      "doi-asserted-by": "publisher",
      "first-page": "191",
      "DOI": "10.1016/j.jsb.2016.06.007",
      "article-title": "Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging",
      "volume": "197",
      "author": "Hagen",
      "year": "2017",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib28",
      "doi-asserted-by": "publisher",
      "first-page": "307",
      "DOI": "10.1016/j.jsb.2017.02.009",
      "article-title": "Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality",
      "volume": "200",
      "author": "Han",
      "year": "2017",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "volume-title": "Intermolecular and Surface Forces",
      "year": "2011",
      "author": "Israelachvili",
      "key": "bib29"
    },
    {
      "key": "bib30",
      "doi-asserted-by": "publisher",
      "first-page": "68",
      "DOI": "10.1016/j.jsb.2012.04.020",
      "article-title": "Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM",
      "volume": "179",
      "author": "Jain",
      "year": "2012",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib31",
      "doi-asserted-by": "publisher",
      "first-page": "254",
      "DOI": "10.1126/science.1138248",
      "article-title": "Structure of fungal fatty acid synthase and implications for iterative substrate shuttling",
      "volume": "316",
      "author": "Jenni",
      "year": "2007",
      "journal-title": "Science"
    },
    {
      "key": "bib32",
      "doi-asserted-by": "publisher",
      "first-page": "12803",
      "DOI": "10.1073/pnas.0805827105",
      "article-title": "Inhibition of the fungal fatty acid synthase type I multienzyme complex",
      "volume": "105",
      "author": "Johansson",
      "year": "2008",
      "journal-title": "PNAS"
    },
    {
      "key": "bib33",
      "doi-asserted-by": "publisher",
      "DOI": "10.15252/msb.20167412",
      "article-title": "Capturing protein communities by structural proteomics in a thermophilic eukaryote",
      "volume": "13",
      "author": "Kastritis",
      "year": "2017",
      "journal-title": "Molecular Systems Biology"
    },
    {
      "key": "bib34",
      "doi-asserted-by": "publisher",
      "first-page": "915",
      "DOI": "10.1126/science.aar7389",
      "article-title": "Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation",
      "volume": "359",
      "author": "Kim",
      "year": "2018",
      "journal-title": "Science"
    },
    {
      "key": "bib35",
      "doi-asserted-by": "publisher",
      "DOI": "10.7554/eLife.18722",
      "article-title": "Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2",
      "volume": "5",
      "author": "Kimanius",
      "year": "2016",
      "journal-title": "eLife"
    },
    {
      "key": "bib36",
      "doi-asserted-by": "publisher",
      "first-page": "71",
      "DOI": "10.1006/jsbi.1996.0013",
      "article-title": "Computer visualization of three-dimensional image data using IMOD",
      "volume": "116",
      "author": "Kremer",
      "year": "1996",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib37",
      "doi-asserted-by": "publisher",
      "first-page": "1443",
      "DOI": "10.1126/science.1251652",
      "article-title": "The resolution revolution",
      "volume": "343",
      "author": "Kuhlbrandt",
      "year": "2014",
      "journal-title": "Science"
    },
    {
      "key": "bib38",
      "doi-asserted-by": "publisher",
      "first-page": "420",
      "DOI": "10.1016/j.jsb.2011.02.005",
      "article-title": "The surface of evaporated carbon films is an insulating, high-bandgap material",
      "volume": "174",
      "author": "Larson",
      "year": "2011",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib39",
      "doi-asserted-by": "publisher",
      "first-page": "319",
      "DOI": "10.1016/j.cell.2007.03.013",
      "article-title": "The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together",
      "volume": "129",
      "author": "Lomakin",
      "year": "2007",
      "journal-title": "Cell"
    },
    {
      "key": "bib40",
      "doi-asserted-by": "publisher",
      "first-page": "36",
      "DOI": "10.1016/j.jsb.2005.07.007",
      "article-title": "Automated electron microscope tomography using robust prediction of specimen movements",
      "volume": "152",
      "author": "Mastronarde",
      "year": "2005",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib41",
      "doi-asserted-by": "publisher",
      "first-page": "1",
      "DOI": "10.1111/j.1365-2818.1983.tb04225.x",
      "article-title": "Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples",
      "volume": "131",
      "author": "McDowall",
      "year": "1983",
      "journal-title": "Journal of Microscopy"
    },
    {
      "key": "bib42",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/s41467-017-00782-3",
      "article-title": "Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy",
      "volume": "8",
      "author": "Naydenova",
      "year": "2017",
      "journal-title": "Nature Communications"
    },
    {
      "key": "bib43",
      "doi-asserted-by": "publisher",
      "first-page": "391",
      "DOI": "10.1021/cr60076a001",
      "article-title": "The Surface Activity of Proteins",
      "volume": "23",
      "author": "Neurath",
      "year": "1938",
      "journal-title": "Chemical Reviews"
    },
    {
      "key": "bib44",
      "doi-asserted-by": "publisher",
      "first-page": "47",
      "DOI": "10.1038/nature14548",
      "article-title": "The architecture of the spliceosomal U4/U6.U5 tri-snRNP",
      "volume": "523",
      "author": "Nguyen",
      "year": "2015",
      "journal-title": "Nature"
    },
    {
      "key": "bib45",
      "doi-asserted-by": "publisher",
      "DOI": "10.7554/eLife.34257",
      "article-title": "Routine single particle CryoEM sample and grid characterization by tomography",
      "volume": "7",
      "author": "Noble",
      "year": "2018",
      "journal-title": "eLife"
    },
    {
      "key": "bib46",
      "doi-asserted-by": "publisher",
      "first-page": "1377",
      "DOI": "10.1073/pnas.63.4.1377",
      "article-title": "Crystallization of a multienzyme complex: fatty acid synthetase from yeast",
      "volume": "63",
      "author": "Oesterhelt",
      "year": "1969",
      "journal-title": "PNAS"
    },
    {
      "key": "bib47",
      "doi-asserted-by": "publisher",
      "first-page": "152",
      "DOI": "10.1016/j.jsb.2009.12.020",
      "article-title": "Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples",
      "volume": "170",
      "author": "Pantelic",
      "year": "2010",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib48",
      "doi-asserted-by": "publisher",
      "first-page": "1375",
      "DOI": "10.1016/j.ssc.2012.04.038",
      "article-title": "The application of graphene as a sample support in transmission electron microscopy",
      "volume": "152",
      "author": "Pantelic",
      "year": "2012",
      "journal-title": "Solid State Communications"
    },
    {
      "key": "bib49",
      "doi-asserted-by": "publisher",
      "DOI": "10.1063/1.4870531",
      "article-title": "Rendering graphene supports hydrophilic with non-covalent aromatic functionalization for transmission electron microscopy",
      "volume": "104",
      "author": "Pantelic",
      "year": "2014",
      "journal-title": "Applied Physics Letters"
    },
    {
      "key": "bib50",
      "doi-asserted-by": "publisher",
      "first-page": "51",
      "DOI": "10.1016/bs.mie.2016.04.011",
      "article-title": "Specimen preparation for High-Resolution Cryo-EM",
      "volume": "579",
      "author": "Passmore",
      "year": "2016",
      "journal-title": "Methods in Enzymology"
    },
    {
      "key": "bib51",
      "doi-asserted-by": "publisher",
      "first-page": "1605",
      "DOI": "10.1002/jcc.20084",
      "article-title": "UCSF chimera--a visualization system for exploratory research and analysis",
      "volume": "25",
      "author": "Pettersen",
      "year": "2004",
      "journal-title": "Journal of Computational Chemistry"
    },
    {
      "key": "bib52",
      "doi-asserted-by": "publisher",
      "first-page": "737",
      "DOI": "10.1146/annurev.biochem.052208.114057",
      "article-title": "Amphipols, Nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions",
      "volume": "79",
      "author": "Popot",
      "year": "2010",
      "journal-title": "Annual Review of Biochemistry"
    },
    {
      "key": "bib53",
      "doi-asserted-by": "publisher",
      "first-page": "41",
      "DOI": "10.1017/S0033583500002900",
      "article-title": "Experimental methods for investigating protein adsorption kinetics at surfaces",
      "volume": "27",
      "author": "Ramsden",
      "year": "1994",
      "journal-title": "Quarterly Reviews of Biophysics"
    },
    {
      "key": "bib54",
      "doi-asserted-by": "publisher",
      "first-page": "216",
      "DOI": "10.1016/j.jsb.2015.08.008",
      "article-title": "CTFFIND4: fast and accurate defocus estimation from electron micrographs",
      "volume": "192",
      "author": "Rohou",
      "year": "2015",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib55",
      "doi-asserted-by": "publisher",
      "first-page": "649",
      "DOI": "10.1038/nmeth.2931",
      "article-title": "Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas",
      "volume": "11",
      "author": "Russo",
      "year": "2014",
      "journal-title": "Nature Methods"
    },
    {
      "key": "bib56",
      "doi-asserted-by": "publisher",
      "first-page": "531",
      "DOI": "10.1016/j.jsb.2013.04.014",
      "article-title": "Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging",
      "volume": "183",
      "author": "Sader",
      "year": "2013",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib57",
      "doi-asserted-by": "publisher",
      "first-page": "14448",
      "DOI": "10.1074/jbc.M116.724153",
      "article-title": "Topology and structure/Function correlation of ring- and Gate-forming domains in the dynamic secretin complex of thermus thermophilus",
      "volume": "291",
      "author": "Salzer",
      "year": "2016",
      "journal-title": "Journal of Biological Chemistry"
    },
    {
      "key": "bib58",
      "doi-asserted-by": "publisher",
      "first-page": "122",
      "DOI": "10.1038/nature26153",
      "article-title": "Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis",
      "volume": "556",
      "author": "Scapin",
      "year": "2018",
      "journal-title": "Nature"
    },
    {
      "key": "bib59",
      "doi-asserted-by": "publisher",
      "first-page": "853",
      "DOI": "10.1038/nmeth.2115",
      "article-title": "Prevention of overfitting in cryo-EM structure determination",
      "volume": "9",
      "author": "Scheres",
      "year": "2012",
      "journal-title": "Nature Methods"
    },
    {
      "key": "bib60",
      "doi-asserted-by": "publisher",
      "first-page": "1192",
      "DOI": "10.1126/science.1199358",
      "article-title": "Three-dimensional model of salmonella's needle complex at subnanometer resolution",
      "volume": "331",
      "author": "Schraidt",
      "year": "2011",
      "journal-title": "Science"
    },
    {
      "key": "bib61",
      "doi-asserted-by": "publisher",
      "DOI": "10.1098/rsob.170255",
      "article-title": "Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence",
      "volume": "8",
      "author": "Takizawa",
      "year": "2018",
      "journal-title": "Open Biology"
    },
    {
      "key": "bib62",
      "doi-asserted-by": "publisher",
      "first-page": "38",
      "DOI": "10.1016/j.jsb.2006.05.009",
      "article-title": "EMAN2: an extensible image processing suite for electron microscopy",
      "volume": "157",
      "author": "Tang",
      "year": "2007",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib63",
      "doi-asserted-by": "publisher",
      "first-page": "214",
      "DOI": "10.1016/j.jsb.2008.06.004",
      "article-title": "Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future",
      "volume": "163",
      "author": "Taylor",
      "year": "2008",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib64",
      "doi-asserted-by": "publisher",
      "first-page": "1",
      "DOI": "10.1016/0095-8522(60)90002-7",
      "article-title": "A theory and method for the spreading of protein monolayers",
      "volume": "15",
      "author": "Trurnit",
      "year": "1960",
      "journal-title": "Journal of Colloid Science"
    },
    {
      "key": "bib65",
      "doi-asserted-by": "publisher",
      "first-page": "170",
      "DOI": "10.1016/j.jsb.2018.01.001",
      "article-title": "Optimizing \"self-wicking\" nanowire grids",
      "volume": "202",
      "author": "Wei",
      "year": "2018",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib66",
      "doi-asserted-by": "publisher",
      "first-page": "189",
      "DOI": "10.1111/j.1432-1033.1979.tb12885.x",
      "article-title": "Studies on the multi-enzyme complex of yeast fatty-acid synthetase. reversible dissociation and isolation of two polypeptide chains",
      "volume": "94",
      "author": "Wieland",
      "year": "1979",
      "journal-title": "European Journal of Biochemistry"
    },
    {
      "key": "bib67",
      "doi-asserted-by": "publisher",
      "first-page": "857",
      "DOI": "10.1038/s41591-018-0042-6",
      "article-title": "Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1",
      "volume": "24",
      "author": "Xu",
      "year": "2018",
      "journal-title": "Nature Medicine"
    },
    {
      "key": "bib68",
      "doi-asserted-by": "publisher",
      "first-page": "470",
      "DOI": "10.1016/j.cell.2017.06.039",
      "article-title": "Structure of the Nav1.4-\u03b21 complex from electric eel",
      "volume": "170",
      "author": "Yan",
      "year": "2017",
      "journal-title": "Cell"
    },
    {
      "key": "bib69",
      "doi-asserted-by": "publisher",
      "first-page": "3290",
      "DOI": "10.1021/la00021a062",
      "article-title": "Two-Dimensional protein array growth in thin layers of protein solution on aqueous subphases",
      "volume": "10",
      "author": "Yoshimura",
      "year": "1994",
      "journal-title": "Langmuir"
    },
    {
      "key": "bib70",
      "doi-asserted-by": "publisher",
      "first-page": "1",
      "DOI": "10.1016/j.jsb.2015.11.003",
      "article-title": "Gctf: real-time CTF determination and correction",
      "volume": "193",
      "author": "Zhang",
      "year": "2016",
      "journal-title": "Journal of Structural Biology"
    },
    {
      "key": "bib71",
      "doi-asserted-by": "publisher",
      "first-page": "331",
      "DOI": "10.1038/nmeth.4193",
      "article-title": "MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy",
      "volume": "14",
      "author": "Zheng",
      "year": "2017",
      "journal-title": "Nature Methods"
    },
    {
      "key": "bib72",
      "doi-asserted-by": "publisher",
      "first-page": "67",
      "DOI": "10.1038/s41586-018-0255-3",
      "article-title": "Structure of a human synaptic GABAA receptor",
      "volume": "559",
      "author": "Zhu",
      "year": "2018",
      "journal-title": "Nature"
    }
  ],
  "container-title": "eLife",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://cdn.elifesciences.org/articles/42747/elife-42747-v2.pdf",
      "content-type": "application/pdf",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://cdn.elifesciences.org/articles/42747/elife-42747-v2.xml",
      "content-type": "application/xml",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://elifesciences.org/articles/42747",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        10,
        12
      ]
    ],
    "date-time": "2023-10-12T00:52:56Z",
    "timestamp": 1697071976000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://elifesciences.org/articles/42747"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2019,
        4,
        1
      ]
    ]
  },
  "references-count": 72,
  "alternative-id": [
    "10.7554/eLife.42747"
  ],
  "URL": "http://dx.doi.org/10.7554/eLife.42747",
  "relation": {
    "is-supplemented-by": [
      {
        "id-type": "accession",
        "id": "EMD-0178",
        "asserted-by": "subject"
      },
      {
        "id-type": "accession",
        "id": "EMD-0179",
        "asserted-by": "subject"
      }
    ],
    "has-review": [
      {
        "id-type": "doi",
        "id": "10.3410/f.735431011.793558538",
        "asserted-by": "object"
      },
      {
        "id-type": "doi",
        "id": "10.7554/eLife.42747.030",
        "asserted-by": "object"
      },
      {
        "id-type": "doi",
        "id": "10.3410/f.735431011.793558933",
        "asserted-by": "object"
      },
      {
        "id-type": "doi",
        "id": "10.7554/eLife.42747.031",
        "asserted-by": "object"
      }
    ],
    "has-preprint": [
      {
        "id-type": "doi",
        "id": "10.1101/400432",
        "asserted-by": "object"
      }
    ]
  },
  "ISSN": [
    "2050-084X"
  ],
  "subject": [],
  "published": {
    "date-parts": [
      [
        2019,
        4,
        1
      ]
    ]
  },
  "article-number": "e42747"
}