Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Gyobu, Nobuhiko; Tani, Kazuhide; Hiroaki, Yoko; Kamegawa, Akiko; Mitsuoka, Kaoru; Fujiyoshi, Yoshinori

doi:10.1016/j.jsb.2004.01.012

Cited by 104 publications

(96 citation statements)

References 20 publications

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“…Specimens for cryo-electron microscopy were prepared according to 13 . Briefly, double-layered AQP0 2D crystals were mixed with an equal amount of 10% trehalose and the suspension applied to a molybdenum grid covered with a thin carbon film.…”

Section: Electron Microscopy and Data Processingmentioning

confidence: 99%

“…Using a better batch of 2D crystals (Fig. 1a), the carbon sandwich specimen preparation technique 13 , and a helium-cooled top-entry 300 kV electron microscope 14 , equipped with a field emission gun and a 4K x 4K charge-coupled device (CCD) camera, we have now been able to collect electron diffraction data to much higher resolution.…”

Section: The 19 å Structure Of Junctional Aqp0mentioning

confidence: 99%

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Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Gonen

Cheng

Sliz

et al. 2005

Nature

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622

644

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Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. We describe a 1.9 Å 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 N-and C-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 could therefore build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions. KeywordsAquaporin-0; lens; MIP; two-dimensional crystal; lipid-protein interaction; electron crystallography Members of the aquaporin (AQP) family form membrane pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol and urea (aquaglyceroporins) (reviewed in 1 ). Structural studies have revealed that all AQPs share the same basic architecture, which consists of two tandem repeats, each containing a bundle of three transmembrane α-helices and a hydrophobic loop with the highly conserved asparagine-proline-alanine (NPA) motif 2 -8 . The two NPA-containing loops B and E fold back into the membrane and form short α-helices (HB and HE) that line the water pore. The ar/R constriction site, so named because it is formed by an aromatic and an arginine residue, confers water selectivity to AQP pores, while the NPA motifs play an important role in the proton exclusion mechanism (reviewed in 9 ).Correspondence to: Thomas Walz.Correspondence and requests for materials should be addressed to T.W. (twalz@hms.harvard.edu). Coordinates and structure factors for junctional and non-junctional AQP0 have been deposited in the Protein Data Bank (accession codes 2B6O and 2B6P, respectively).. Suplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. AQP0 is the most abundant protein in lens fibre cell membranes, where it forms not only water pores but also the 11-13 nm "thin lens junctions" that assemble upon proteolytic cleavage of the cytoplasmic termini 10 , 11 . We recently presented the structure of the AQP0-mediated membrane junction at 3 Å resolution as determined by electron crystallography of doublelayered two-dimensional (2D) crystals 7 . The structure showed that AQP0 junctions are stabilised by specific interactions between tetramers in adjoining membranes involving almost exclusively proline residues. Calculated pore profiles also showed that the pore in junctional AQP0 is highly constricted due to a substantially ...

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Section: Electron Microscopy and Data Processingmentioning

confidence: 99%

Section: The 19 å Structure Of Junctional Aqp0mentioning

confidence: 99%

Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Gonen

Cheng

Sliz

et al. 2005

Nature

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622

644

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“…Samples were negatively stained with 2% (wt/vol) uranyl acetate to screen for crystallization conditions. Specimens for cryo-EM were prepared in a cold room using the carbon sandwich method (34).…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Abe

Tani²,

Friedrich

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Gastric H + ,K + -ATPase is responsible for gastric acid secretion. ATPdriven H + uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K + , resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H + /2K + to 1H + /1K + per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K + congener (Rb + ), the 8-Å resolution structure of H + ,K + -ATPase in the transition state of dephosphorylation, (Rb + )E2∼AlF, which is distinct from the preceding Rb + -free E2P state. A strong density located in the transmembrane cation-binding site of (Rb + )E2∼AlF highly likely represents a single bound Rb + ion, which is clearly different from the Rb + -free E2AlF or K + -bound (K + )E2∼AlF structures. Measurement of radioactive 86 Rb + binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb + is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H + /1K + /1ATP transport mode of H + ,K + -ATPase, which is a prerequisite for generation of the 10 6 -fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb + )E2∼AlF structure, we propose a refined vectorial transport model of H + ,K + -ATPase, which must prevail against the highly acidic state of the gastric lumen.electron crystallography | P-type ATPases | membrane proteins | bioenergetics L ike other P-type ATPases (1), the vectorial cation transport of gastric H + ,K + -ATPase (2) is accomplished by cyclical conformational changes of the enzyme (abbreviated as E), generally described using an E1/E2 nomenclature based on the PostAlbers scheme for Na + ,K + -ATPase (3) (SI Appendix, Fig. S1). In contrast to the closely related Na + ,K + -ATPase, which exchanges three Na + for two K + ions in an electrogenic transport reaction, gastric H + ,K + -ATPase operates electroneutrally, although its transport stoichiometry (2H + /2K + /ATP or 1H + /1K + /ATP) has remained controversial (4, 5). A measurement of the proton transport (5) revealed an H + /ATP ratio of 2 at neutral pH. Accordingly, two K + ions must be counter transported during a single turnover of the transport cycle, accompanied by the hydrolysis of one ATP molecule (2H + /2K + /ATP). The reported free energy for ATP hydrolysis of the gastric secretory membrane [−13 kcal/mol (4)] provides sufficient energy to achieve a maximum change in pH (ΔpH) of 4.7 units when two H + ions are transported per hydrolysis of one ATP molecule; thus, the generation of pH 1 (ΔpH > 6 units in the stomach) with a 2H + /2K + / ATP stoichiometry is thermodynamically impossible. Therefore,...

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“…To further analyze the packing of the channels in the membranes, PAP channel-containing vesicles produced at an mCLR of 0.909 were prepared by the carbon sandwich technique (47) and analyzed by cryo-EM (Fig. 4B, Left).…”

Section: Simulationsmentioning

confidence: 99%

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Shen

Wen

Erbakan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

165

246

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Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar [5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10 −14 cm 3 /s or 3.5(±1.0) × 10 8 water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10 8 water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10 8 water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10 7 water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10 5 pores per μm 2 ) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10 3 pores per μm 2 ). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.artificial aquaporins | artificial water channels | peptide-appended pillar [5]arene | single-channel water permeability | two-dimensional arrays T he discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1-9). Areas of application include liquid and gas separations (10-13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen-Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The largescale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water chan...

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Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Cited by 104 publications

References 20 publications

Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

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Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Cited by 104 publications

References 20 publications

Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Contact Info

Product

Resources

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Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site