Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data

Abstract

The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.

Figure 1. XFEL data analysis.

(a) Distribution of Ewald sphere wavelengths for all reflections on a single image, before (grey, thick) and after (black, thin) orientation matrix refinement. Refinement resulted in a 0.117° rotational shift in the orientation model. (b) Cluster algorithm by a modified version of algorithm of Brehm and Diederichs, showing positions of final artificially defined vectors corresponding to individual images (see Methods). Note the vectors fall into two sets, clearly separating the two indexing choices.

Figure 2. Structure of CPV17 polyhedrin.

(a) Electron density around the ATP moiety contoured at 1.3 σ. (b) Cartoon of the CPV17 polyhedrin subunit coloured from blue to red, N terminus to C terminus that are denoted by spheres. Secondary structure elements are labelled and the ATP molecule shown as sticks. (c) Comparison of the CPV17 and CPV1 polyhedrin structures. The molecules were aligned with program SHP. Both colour and tube thickness represent r.m.s. distance (r.m.s.d.) of equivalent C-alpha atoms (thin, blue: r.m.s.d.<1.0 Å, green, thicker: 1.0–2.5 Å, orange, thickest: >2.5 Å). Unaligned regions are coloured red and displayed with exaggerated thickness. Variable regions are labelled v1–v5 with the N- and C-terminal extensions of CPV1 drawn. (d) Comparison of variable regions between CPV17 (magenta) and CPV1 (cyan). (e) The interface between monomers (coloured differently) in the crystal is rich in tyrosines.

Figure 3. Disulphide bonds link chains of CPV17 polyhedrin molecules.

(a) Electron density around C142 (pale green) and C155 (pink) from a related molecule in the crystal contoured at 1.0 σ for CPV17 at 293 K. (b) SDS–polyacrylamide gel electrophoresis analysis of CPV17 crystals with and without reducing agent 2-mercapto-ethanol. (c) Visualization of the intermolecular disulphide-linked helical strings going through the crystal lattice of CPV17. Four helical strings are drawn, one with individual polyhedrin molecules coloured separately, the others coloured pink, cream and blue. (d) Electron density around C142 (pale green) and C155 (pink) from a related molecule in the crystal contoured at 1.0 σ for CPV17 at 100 K.

Figure 4. ATP interactions.

(a) Interactions of the base moiety of ATP of CPV17. Arg 8 (coloured green) is from a related molecule. Alignment of the ATP molecule for (b) CPV17 and (c) CPV1.