Ultrastable gold substrates for electron cryomicroscopy

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Science  12 Dec 2014:
Vol. 346, Issue 6215, pp. 1377-1380
DOI: 10.1126/science.1259530

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  1. Fig. 1 Ultrastable substrate design.

    The ultrastable gold support comprises a 3-mm diameter disc of gold mesh (A) where a Embedded Image Å thick layer of gold foil with a regular array of micrometer-sized holes is suspended across the square openings in the mesh (diagramed along the section indicated). After application of an aqueous protein sample and plunge freezing at Embedded Image K, each hole contains a thin film of protein particles embedded in vitrified ice. (A to C) Optical micrographs of the gold grid at low, medium, and high magnification respectively; each hole is 1.2 μm in diameter and sets the scale for (B) to (D). (D) Transmission electron micrograph of an individual 1.2-μm hole containing vitrified ice. (E) A typical high-resolution electron micrograph of apoferritin suspended in ice on a gold grid. Scale bars for the micrograph and magnified inset are 1200 and 120 Å, respectively.

  2. Fig. 2 Reduced motion of gold substrates under high-energy electron irradiation.

    (A) Measurements of the vertical motion of am-C and gold supports under typical cryo-EM illumination conditions (300 keV, 16 e2 per second, and 80 K). Each point is the magnitude of vertical displacement of the edge of a particular hole in the foil relative to its initial position before electron irradiation. Each solid line is the root mean square displacement for multiple holes in multiple squares of one grid. When a typical thin layer of vitreous ice is present (B), the vertical motion of both substrates is reduced about twofold, and the motion on am-C becomes more complicated in nature.

  3. Fig. 3 Reduced particle motion and improved resolution on ultrastable grids.

    (A) 80S ribosomes were tracked during electron irradiation (using the same imaging conditions) for particles supported on three different am-C grids [black circles (17)] and three different gold grids (gold crosses). Each point is the in-plane ensemble average displacement of a particle from its initial position, and the error bars denote the SEM for the multiple grids tested. RMS, root mean square. Solid lines are linear fits to the two phases of motion, where the slopes are the average speeds of the particles. There was a 43% reduction in the speed of the first phase (8.8 to 5.0 Å/s) and a 77% reduction in the speed of the second phase (2.3 to 0.53 Å/s) on gold relative to am-C. Density maps of apoferritin processed identically from data collected using identical conditions for am-C (B) and gold (C) substrates. The map in (B) has a resolution of 25 Å (gold-standard Fourier shell correlation) and contains no discernible information beyond that present in the initial model. Compare to (C) with a resolution of 8.0 Å, which resolves the entire molecular chain.

  4. Fig. 4 The structure of apoferritin from 483 single-particle images.

    (A) The previously published x-ray crystal structure is fit to the cryo-EM density map, and the density is cut in half along the plane defined by two of the fourfold symmetry axes to reveal the interior of the complex. The cut surface shows the local resolution of the density using the color scale as indicated. (B) View of one of the α helices showing clear side-chain density. (C) View along the twofold symmetry axis showing clear separation between the β strands. (D) Section parallel to the fourfold pore axis showing the corrugated density of the residues in the interior of the pore.

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