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  1. Article ; Online: Devitrification reduces beam-induced movement in cryo-EM

    Jan-Philip Wieferig / Deryck J. Mills / Werner Kühlbrandt

    IUCrJ, Vol 8, Iss 2, Pp 186-

    2021  Volume 194

    Abstract: As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent ... ...

    Abstract As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å−2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.
    Keywords cryo-em ; devitrification ; beam-induced movement ; Science ; Q
    Language English
    Publishing date 2021-03-01T00:00:00Z
    Publisher International Union of Crystallography
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Author Correction

    Eva S. Cunha / Xiaorui Chen / Marta Sanz-Gaitero / Deryck J. Mills / Hartmut Luecke

    Nature Communications, Vol 14, Iss 1, Pp 1-

    Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution

    2023  Volume 1

    Keywords Science ; Q
    Language English
    Publishing date 2023-02-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution

    Eva S. Cunha / Xiaorui Chen / Marta Sanz-Gaitero / Deryck J. Mills / Hartmut Luecke

    Nature Communications, Vol 12, Iss 1, Pp 1-

    2021  Volume 8

    Abstract: Infection by Helicobacter pylori is associated with peptic ulcers and gastric cancer. H. pylori urease is required for colonization of the stomach and thus an attractive antimicrobial drug target. Cryo-EM analyses of the H. pylori urease with inhibitors ... ...

    Abstract Infection by Helicobacter pylori is associated with peptic ulcers and gastric cancer. H. pylori urease is required for colonization of the stomach and thus an attractive antimicrobial drug target. Cryo-EM analyses of the H. pylori urease with inhibitors bound reveal structural details useful in rational drug design.
    Keywords Science ; Q
    Language English
    Publishing date 2021-01-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria

    Yongchan Lee / Pattama Wiriyasermkul / Pornparn Kongpracha / Satomi Moriyama / Deryck J. Mills / Werner Kühlbrandt / Shushi Nagamori

    Nature Communications, Vol 13, Iss 1, Pp 1-

    2022  Volume 19

    Abstract: Cystinuria is caused by mutations in heterodimeric amino acid transporter known as system b0,+. Here, authors discover that Ca2+ stabilizes the interface between two system b0,+ regulatory subunits rBAT, leading to super-dimerization of the b0,+AT–rBAT ... ...

    Abstract Cystinuria is caused by mutations in heterodimeric amino acid transporter known as system b0,+. Here, authors discover that Ca2+ stabilizes the interface between two system b0,+ regulatory subunits rBAT, leading to super-dimerization of the b0,+AT–rBAT heterodimer, facilitating system b0,+ maturation.
    Keywords Science ; Q
    Language English
    Publishing date 2022-05-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: A comparative study of single-particle cryo-EM with liquid-nitrogen and liquid-helium cooling

    Olivia Pfeil-Gardiner / Deryck J. Mills / Janet Vonck / Werner Kuehlbrandt

    IUCrJ, Vol 6, Iss 6, Pp 1099-

    2019  Volume 1105

    Abstract: Radiation damage is the most fundamental limitation for achieving high resolution in electron cryo-microscopy (cryo-EM) of biological samples. The effects of radiation damage are reduced by liquid-helium cooling, although the use of liquid helium is more ...

    Abstract Radiation damage is the most fundamental limitation for achieving high resolution in electron cryo-microscopy (cryo-EM) of biological samples. The effects of radiation damage are reduced by liquid-helium cooling, although the use of liquid helium is more challenging than that of liquid nitrogen. To date, the benefits of liquid-nitrogen and liquid-helium cooling for single-particle cryo-EM have not been compared quantitatively. With recent technical and computational advances in cryo-EM image recording and processing, such a comparison now seems timely. This study aims to evaluate the relative merits of liquid-helium cooling in present-day single-particle analysis, taking advantage of direct electron detectors. Two data sets for recombinant mouse heavy-chain apoferritin cooled with liquid-nitrogen or liquid-helium to 85 or 17 K were collected, processed and compared. No improvement in terms of resolution or Coulomb potential map quality was found for liquid-helium cooling. Interestingly, beam-induced motion was found to be significantly higher with liquid-helium cooling, especially within the most valuable first few frames of an exposure, thus counteracting any potential benefit of better cryoprotection that liquid-helium cooling may offer for single-particle cryo-EM.
    Keywords cryo-em ; electron cryo-microscopy ; radiation damage ; helium ; liquid-helium cooling ; beam-induced motion ; beam-induced movement ; apoferritin ; Science ; Q
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher International Union of Crystallography
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy.

    Janet Vonck / David N Parcej / Deryck J Mills

    PLoS ONE, Vol 11, Iss 7, p e

    2016  Volume 0159476

    Abstract: The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD ... ...

    Abstract The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.
    Keywords Medicine ; R ; Science ; Q
    Subject code 500 ; 540
    Language English
    Publishing date 2016-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Functional asymmetry and electron flow in the bovine respirasome

    Joana S Sousa / Deryck J Mills / Janet Vonck / Werner Kühlbrandt

    eLife, Vol

    2016  Volume 5

    Abstract: Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most ... ...

    Abstract Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.
    Keywords Bos taurus ; cryo-EM ; mitochondria ; respiratory chain ; supercomplex ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 500
    Language English
    Publishing date 2016-11-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Structural basis of proton translocation and force generation in mitochondrial ATP synthase

    Niklas Klusch / Bonnie J Murphy / Deryck J Mills / Özkan Yildiz / Werner Kühlbrandt

    eLife, Vol

    2017  Volume 6

    Abstract: ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact ... ...

    Abstract ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.
    Keywords ATP synthase ; electron cryo-microscopy ; membrane protein structure ; mitochondria ; membrane potential ; energy conversion ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 500
    Language English
    Publishing date 2017-12-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article: Cryo-EM Structure of the TOM Core Complex from Neurospora crassa

    Bausewein, Thomas / Beate Nitschke / Deryck J. Mills / Julian D. Langer / Stephan Nussberger / Werner Kühlbrandt

    Cell. 2017 Aug. 10, v. 170

    2017  

    Abstract: The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane ... ...

    Abstract The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery.
    Keywords cell membranes ; cryo-electron microscopy ; cytosol ; imports ; membrane proteins ; mitochondria ; Neurospora crassa ; protein subunits
    Language English
    Dates of publication 2017-0810
    Size p. 693-700.e7.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2017.07.012
    Database NAL-Catalogue (AGRICOLA)

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  10. Article ; Online: Cryo-EM structure of respiratory complex I at work

    Kristian Parey / Ulrich Brandt / Hao Xie / Deryck J Mills / Karin Siegmund / Janet Vonck / Werner Kühlbrandt / Volker Zickermann

    eLife, Vol

    2018  Volume 7

    Abstract: Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from ... ...

    Abstract Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
    Keywords respiratory complex I ; redox-linked proton translocation ; active/deactive transition ; Yarrowia lipolytica ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2018-10-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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