Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438, 633-638

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Nature (Impact Factor: 41.46). 01/2006; 438(7068):633-8. DOI: 10.1038/nature04321
Source: PubMed

ABSTRACT Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.

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Available from: Yifan Cheng, Sep 26, 2015
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    • "Atomic detail had been first achieved for macromolecular assemblies on 2-D crystalline arrays, and most recently even on pure single particle reconstructions. The history of reaching atomic resolution reads roughly as follows: (a) diffraction data of protein crystals: Taylor and Glaeser 1974 (catalase crystals); (b) 2-D crystals of membrane proteins: Henderson et al., 1990 (bacteriorhodopsin; current world record was achieved with acquaporin-0 at 1.9 Å (Gonen et al. 2005); (c) 2-D crystalline arrays of soluble proteins: Nogales et al. 1998 (tubulin); (d) helical assemblies: Miyazawa et al. 2003 (acetylcholine receptor reconstituted in lipid tubes) and Yonekura et al. 2003 (bacterial flagella); (e) icosahedral particles: Liu et al. 2010 (adenovirus); (f) single-particle reconstructions: Li et al. 2013 (proteasome). From noisy cryo-EM 2-D projections to atomic-detail 3-D reconstructions Averaging over hundreds and thousands of identical image elements is an incredibly powerful method to reduce electron microscopy-based image noise, by now often far enough to reveal atomic resolution structural detail. "
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    ABSTRACT: Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.
    Protoplasma 01/2014; 251(2). DOI:10.1007/s00709-013-0600-1 · 2.65 Impact Factor
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    • "The second example is an avian mitochondrial complex II (2H88:C) [41], where its TMH3 (curved) shares 13 contact pairs with TMH2. Lastly, in the structure of aquaporin-0 (2B6O:A) [42], its TMH6 (linear) associates with TMH4 via a dense cluster of 16 contacts. Based on the above findings, regular TMHs with strong and constrained interhelical interactions in a single helical interface reveal periodicity in rASA of their residues, thus making rotational angle prediction highly accurate. "
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    ABSTRACT: Since membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle. Here, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32 . More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05 , which is better than 71.47 obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew's correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at TMexpo can better predict rASA and rotational angles than the compared methods. When rotational angles can be accurately predicted, free modeling of transmembrane protein structures in turn may benefit from a reduced complexity in ensembles with a significantly less number of packing arrangements. Furthermore, sequence-based prediction of both rotational angle and lipid exposure can provide essential information when high-resolution structures are unavailable and contribute to experimental design to elucidate transmembrane protein functions.
    BMC Bioinformatics 10/2013; 14(1):304. DOI:10.1186/1471-2105-14-304 · 2.58 Impact Factor
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    • "In highly ordered two-dimensional crystals of membrane proteins (diffracting to resolution better than 3.5 Å) protein–protein contacts are often absent as well. The examples are 2D crystals of bR (Grigorieff et al., 1996; Mitsuoka et al., 1999), aquaporin (Gonen et al., 2005; Hite et al., 2010) and, as we can deduce now from our crystallographic structures, in OmpF (Sass et al., 1989b). The space between protein molecules in these cases is filled with completely or partially ordered lipids indicating that interaction of protein with annular lipids is an important factor determining the crystalline order. "
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    ABSTRACT: Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a "membrane lyzozyme". The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein-protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.
    Journal of Structural Biology 03/2012; 178(3):311-8. DOI:10.1016/j.jsb.2012.03.005 · 3.23 Impact Factor
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