1. Tricorn Protease Exists as an Icosahedral Supermolecule In Vivo
Jochen Walz, Tomohiro Tamura, Noriko Tamura, Rudo Grimm, Wolfgang Baumeister, Abraham J Koster 
Molecular Cell 
Volume 1, Issue 1, Pages (December 1997)
DOI: /S (00)

2. Figure 1
Elution Profile of Tricorn Protease and Electron Micrograph of the Void Volume Fraction after Superose 6 Chromatography
(A) A sample containing 300 μg of isolated tricorn protease was fractionated by Superose 6 molecular sieve chromatography. Protein was detected by absorbance at 280 nm. Samples (10 μl each) from several fractions were subjected to SDS–PAGE (top panel). Bands migrating to the same positions were detected in the void volume fraction and in the 730 kDa fraction, which corresponds to the tricorn hexamer. Tricorn peptidase activity was detected by incubating 5 μl of each fraction with 10 nmol of H-AAF-AMC at 60°C for 30 min and measuring released AMC fluorometrically.
(B) The void volume fractions were subjected to electron microscopy after negative staining with uranyl acetate. The prominent spherical structures, 55 nm in diameter, represent the tricorn capsids. Because of the fragile nature of the capsids, they tend to disassemble into hexameric units seen in the background. Scale bar in (B), 200 nm.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

3. Figure 2
Montage of Electron Micrographs of Frozen Hydrated Tricorn Capsids
(A) was recorded close to focus (0.5 μm underfocus) and contains higher resolution information, with lower contrast, compared to (B), which was recorded at an underfocus of ∼5 μm. (C) shows the zero tilt view of a tomographic tilt series containing two capsid structures (∼7 μm underfocus). The micrograph shown in (C) is exposed with a 10-fold lower electron dose than (B) (1.5–2 e−/Å2 compared to 15–20 e−/Å2). The four small, dark, circular features in the image are gold beads used as fiducial markers in the tomographic reconstruction. Scale bars in (A)–(C), 50 nm.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

4. Figure 3
Reconstruction of the Tricorn Capsid from Tomographic Tilt Series
(A) A gallery of 1.26 nm thick slices through a single reconstructed tricorn capsid. The tricorn capsid particle is hollow and does not contain any discernible features inside. In spite of the low signal-to-noise ratio in the individual 2-D slices, an indication of five-fold symmetry, most apparent in the third row from the top, can be observed.
(B) Surface representation of the tricorn capsid after averaging seven individually reconstructed particles were averaged but without imposing icosahedral symmetry. The view is down the five-fold symmetry axis.
(C–E) Views of the reconstructed capsid after icosahedral symmetrization down the three-fold axis, five-fold axis, and two-fold axis, respectively. Scale bars in (A)–(E), 50 nm.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

5. Figure 4 Resolution Assessment
Resolution assessment of the tomographic reconstruction (dotted line) and the icosahedral reconstruction (solid line) using the Fourier shell correlation function. The 23.2 σ curve (dashed line) is used as criterion for the resolution limit.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

6. Figure 5 Flow Diagram of Image Processing
Flow chart of the basic steps involved the 3-D map reconstruction of the capsid shown in Figure 6.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

7. Figure 6 Reconstruction of the Tricorn Capsid from 2-D Projections
(A) A surface representation of the tricorn capsid at 2.5 nm resolution.
(B) Top: end-on view of a single tricorn hexameric toroid taken out of the capsid and cut open horizontally; bottom: hexameric toroid cut open vertically. The large internal cavity is clearly visible. Scale bar in (A), 50 nm; in (B), 15 nm.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )

8. Figure 7
Electron Cryomicroscopy of Partially Lysed T. acidophilum Cells
(A) Electron micrograph of a lysed T. acidophilum cell. The opening of the cell and a stream of cellular contents leaking out are visible.
(B) Image of a similar “ghost cell” at higher magnification. The arrows point to several dense structures in a size range corresponding to tricorn capsids. Scale bar in (A), 200 nm; in (B), 100 nm.
Molecular Cell 1997 1, 59-65DOI: ( /S (00) )