Crystal structure of small protein crambin at 0.48 Å resolution

Abstract

With the development of highly brilliant and extremely intense synchrotron X-ray sources, extreme high-resolution limits for biological samples are now becoming attainable. Here, a study is presented that sets the record in crystallographic resolution for a biological macromolecule. The structure of the small protein crambin was determined to 0.48 Å resolution on the PETRA II ring before its conversion to a dedicated synchrotron-radiation source. The results reveal a wealth of details in electron density and demonstrate the possibilities that are potentially offered by a high-energy source. The question now arises as to what the true limits are in terms of what can be seen at such high resolution. From what can be extrapolated from the results using crystals of crambin, this limit would be at approximately 0.40 Å, which approaches that for smaller compounds.

Figure 1

Sequence alignment of crambin with the only other homologous toxin families purothionin (PDB entry 1bhp), hordothionin (1wuw) and viscotoxin (3c8p). The red arrows indicate the residues that are essential for membrane binding. Yellow, green and blue shading indicates increased homology/identity.

Figure 2

Overlay of crambin (violet) with β-purothionin (PDB entry 1bhp, yellow; Stec et al., 1995 ▶) and β-hordothionin (1wuw, magenta; Johnson et al., 2005 ▶) and their surface-charge representation.

Figure 3

Simulation of increased primary-beam intensity and extrapolation of the resolution limits for crambin from previous data collected on BW7A. The I/σ(I) ratio for the 16 bins within the resolution range 0.62–0.54 Å for the earlier data is shown in violet. The expected increase in this ratio at the PETRA1 beamline assuming a 1000-fold increase in intensity is shown in dark blue. A value of 1 on the vertical axis corresponds to I/σ(I) = 2.8, which was obtained for the outer shell in the earlier 0.54 Å data set. A linear extrapolation of the blue dependence gives a value of 1 at a resolution of just over 6 Å⁻², which corresponds to about 0.4 Å.

Figure 4

Wilson plots for the 0.48 and 0.54 Å (PDB entry 1ejg) data sets.

Figure 5

Diffraction image with the detector set at 45° inclination. The ovals are traces of ice rings; the outermost overloads are at 1 Å resolution. The top edge of the detector is at a resolution of 0.40 Å.

Figure 6

The structure of crambin. (a) Rainbow-coloured ribbon representation starting in blue at the N-terminus; (b) surface charges.

Figure 7

Electron density (cyan) around Arg10 calculated at 0.65 Å before refinement with MoPro commenced. The F _o − F _c map is contoured at 2.7σ (0.25 e Å⁻³) above the mean. Bonding electron density is indicated.