In cellulo structure determination of a novel cypovirus polyhedrin

Abstract

This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.

Keywords: data collection; in cellulo; microcrystals; viral protein.

Figure 1

(a) Uninfected cells. (b) Infected cells. (c) Isolated crystals loaded onto a 25 µm mesh, pictured with an on-axis sample view on the beamline during data collection. (d) Infected cells loaded onto a 25 µm mesh, pictured with an on-axis sample view on the beamline during data collection; the inset shows a close-up of a single cell with two crystals visible. All panels are shown on the same scale. Red arrows indicate polyhedra crystals.

Figure 2

Results of a grid scan from in cellulo data collection shown as a contour plot of the DISTL output ‘good Bragg candidates’.

Figure 3

Images of the hk0 central sections of intensity-weighted reciprocal lattices. (a) and (b) show the unreduced indices, whilst (c) and (d) are after applying crystallographic symmetry. (a) and (c) are for the in cellulo merged data set, while (b) and (d) are for the isolated crystal merged data set. The images were generated by ViewHKL (CCP4). The completeness and multiplicity were 91% and 3.7 and 97% and 9.9, respectively.

Figure 4

Representative electron density: (a) in cellulo, (b) isolated. Maps shown are 2F _o − F _c contoured at 1σ.