Feasibility of one-shot-per-crystal structure determination using Laue diffraction

Abstract

Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a 'pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20-30 microm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used.

Figure 1

Diagram of the setup for the reflection and transmission mirrors used to create a tunable large-bandwidth beam.

Figure 2

Predicted and actual spectrum of the 30% bandwidth (FWHM) X-ray beam. Red, calculated reflection from a Rh-coated reflection mirror at 0.28° (4.9 mrad). Blue, calculated transmission through an Si₃N₄ transmission mirror at 0.24° (4.2 mrad). Magenta, calculated spectrum produced by two reflections and one transmission. Black, spectrum measured using Compton scattering from a Kapton foil. The sharp peaks arise from fluorescent scattering from impurities in the foil and are not present in the incident X-ray beam. Green, spectrum determined from Laue data modeled with 15 Chebyshev polynomials. The heights of the black and green curves have been scaled to match the magenta curve.

Figure 3

Laue diffraction image taken from a small lysozyme crystal, with magnified views of two small regions of the image. The divergence from the capillary was too large, resulting in many overlapping diffraction spots. The odd shapes seen in these particular spots represent approximately half of the full far-field pattern (Fig. 5 ▶), with a small portion of the direct beam (owing to pre-capillary beamstop misalignment). If the full divergence from the capillary were used, the diffraction spots would have elliptical shapes.

Figure 4

Laue diffraction from a lysozyme crystal. Left: crystal on a MicroMesh mount. The circle around the crystal is ∼100 µm in diameter; the crystal is about 30 µm across. Right: diffraction pattern from the crystal with a 10 s exposure time. The inset shows well separated acceptably shaped spots.

Figure 5

Images of the capillary-focused beam observed on a fluorescent screen attached to a small video camera located 300–400 mm downstream of the capillary, i.e. well beyond the sample position. These images reveal the angular profile of the beam passing through the ∼13 µm focal spot. (a) Full far-field image with no upstream stop for the central beam. The central spot is from the direct beam passing straight through the capillary; the outer ring is from X-rays that have been focused by a single reflection off the inner wall of the capillary. (b) Slit-down far-field image of a beam suitable for collecting Laue images with well separated spots. The grids in the images have 1 × 1 mm squares.

Figure 6

Difference Fourier maps around residue 41, which is His in turkey lysozyme and Gln in chicken lysozyme. The contour level is 3σ, with green for positive and red for negative peaks. The data sets used were (a) monochromatic ‘complete’, (b) Laue ‘best’, (c) Laue ‘small firsts’, (d) monochromatic ‘small’, (e) Laue ‘firsts’, (f) Laue ‘single-crystal’; see the text for the definitions of these designations. The structure of turkey lysozyme after refinement but before mutations is shown as multicolored sticks; the corresponding part of chicken lysozyme from PDB entry 1bwh is shown as thin blue sticks. Maps and model were displayed using Coot.

Figure 7

Completeness (blue) and R _p.i.m. (red) as a function of resolution for the six data sets described in the text. ×s, monochromatic ‘complete’; crosses, monochromatic ‘small’; open circles, Laue ‘best’; filled circles, Laue ‘firsts’; open squares, Laue ‘single-crystal’; filled squares, Laue ‘small firsts’.