@article {47083, title = {Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0.}, journal = {Journal of Structural Biology}, year = {2015}, month = {2015 Aug 28}, abstract = {

The K2 Summit camera was initially the only commercially available direct electron detection camera that was optimized for high-speed counting of primary electrons and was also the only one that implemented centroiding so that the resolution of the camera can be extended beyond the Nyquist limit set by the physical pixel size. In this study, we used well-characterized two-dimensional crystals of the membrane protein aquaporin-0 to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases.

}, issn = {1095-8657}, doi = {10.1016/j.jsb.2015.08.015}, author = {Chiu, Po-Lin and Li, Xueming and Li, Zongli and Beckett, Brian and Brilot, Axel F and Grigorieff, Nikolaus and Agard, David A and Cheng, Yifan and Walz, Thomas} } @article {46571, title = {A primer to single-particle cryo-electron microscopy}, journal = {Cell }, volume = {161}, year = {2015}, month = {2015 Apr 23}, pages = {438-49}, chapter = {438-49}, abstract = {

Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.

}, doi = {10.1016/j.cell.2015.03.050}, author = {Cheng, Yifan and Grigorieff, Nikolaus and Penczek, Pawel A and Walz, Thomas} } @article {6338, title = {GPU-enabled FREALIGN: accelerating single particle 3D reconstruction and refinement in Fourier space on graphics processors.}, journal = {Journal of Structural Biology}, volume = {172}, year = {2010}, month = {2010 Dec}, pages = {407-12}, abstract = {

Among all the factors that determine the resolution of a 3D reconstruction by single particle electron cryo-microscopy (cryoEM), the number of particle images used in the dataset plays a major role. More images generally yield better resolution, assuming the imaged protein complex is conformationally and compositionally homogeneous. To facilitate processing of very large datasets, we modified the computer program, FREALIGN, to execute the computationally most intensive procedures on Graphics Processing Units (GPUs). Using the modified program, the execution speed increased between 10 and 240-fold depending on the task performed by FREALIGN. Here we report the steps necessary to parallelize critical FREALIGN subroutines and evaluate its performance on computers with multiple GPUs.

}, keywords = {Computer-Assisted, Cryoelectron Microscopy, Image Processing, Imaging, Three-Dimensional}, issn = {1095-8657}, doi = {10.1016/j.jsb.2010.06.010}, author = {Li, Xueming and Grigorieff, Nikolaus and Cheng, Yifan} } @article {45962, title = {Polymorphic assemblies and crystalline arrays of lens tetraspanin MP20.}, journal = {Journal of Molecular Biology}, volume = {376}, year = {2008}, month = {2008 Feb 15}, pages = {380-92}, abstract = {

Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 A resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.

}, keywords = {Animals, Buffers, Cations, Divalent, Cell Membrane, Chelating Agents, Crystallization, Detergents, Edetic Acid, Egtazic Acid, Eye Proteins, Hydrogen-Ion Concentration, Imaging, Three-Dimensional, Lens, Crystalline, Lipid Metabolism, Magnesium, Membrane Microdomains, Membrane Proteins, Protein Structure, Secondary, Protein Structure, Tertiary, Sheep, Silver Staining, Solubility, Tromethamine}, issn = {1089-8638}, doi = {10.1016/j.jmb.2007.09.001}, author = {Gonen, Tamir and Hite, Richard K and Cheng, Yifan and Petre, Benjamin M and Kistler, Joerg and Walz, Thomas} } @article {45957, title = {Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.}, journal = {Nature}, volume = {438}, year = {2005}, month = {2005 Dec 1}, pages = {633-8}, 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.

}, keywords = {Animals, Aquaporins, Crystallization, Crystallography, Hydrogen Bonding, Lens, Crystalline, Lipid Bilayers, Membrane Lipids, Models, Molecular, Protein Binding, Protein Structure, Quaternary, Sheep, Water}, issn = {1476-4687}, doi = {10.1038/nature04321}, author = {Gonen, Tamir and Cheng, Yifan and Sliz, Piotr and Hiroaki, Yoko and Fujiyoshi, Yoshinori and Harrison, Stephen C and Walz, Thomas} } @article {45956, title = {Aquaporin-0 membrane junctions form upon proteolytic cleavage.}, journal = {Journal of Molecular Biology}, volume = {342}, year = {2004}, month = {2004 Sep 24}, pages = {1337-45}, abstract = {

Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.

}, keywords = {Amino Acid Sequence, Animals, Aquaporins, Biopolymers, Eye Proteins, Hydrolysis, Membrane Glycoproteins, Microscopy, Electron, Molecular Sequence Data, Sheep}, issn = {0022-2836}, doi = {10.1016/j.jmb.2004.07.076}, author = {Gonen, Tamir and Cheng, Yifan and Kistler, Joerg and Walz, Thomas} } @article {45955, title = {Aquaporin-0 membrane junctions reveal the structure of a closed water pore.}, journal = {Nature}, volume = {429}, year = {2004}, month = {2004 May 13}, pages = {193-7}, abstract = {

The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.

}, keywords = {Animals, Aquaporins, Biological Transport, Cell Membrane, Cell Membrane Permeability, Cryoelectron Microscopy, Crystallization, Electric Conductivity, Eye Proteins, Hydrogen-Ion Concentration, Lens, Crystalline, Membrane Glycoproteins, Models, Molecular, Molecular Sequence Data, Protein Conformation, Sheep, Water}, issn = {1476-4687}, doi = {10.1038/nature02503}, author = {Gonen, Tamir and Sliz, Piotr and Kistler, Joerg and Cheng, Yifan and Walz, Thomas} }