Small-rotative fixed-target serial synchrotron crystallography (SR-FT-SSX) for molecular crystals

Abstract

The increasing availability of ultrabright Light Sources is facilitating the study of smaller crystals at faster timescales but with an increased risk of severe X-ray damage, leading to developments in multi-crystal methods such as serial crystallography (SX). SX studies on crystals with small unit cells are challenging as very few reflections are recorded in a single data image, making it difficult to determine the orientation matrix for each crystal and thus preventing the combination of the data from all crystals for structure solution. We herein present a Small-Rotative Fixed-Target Serial Synchrotron Crystallography (SR-FT-SSX) methodology, in which rotation of the serial target through a small diffraction angle $\left(\phi \right)$ at each crystal delivers high-quality data, facilitating ab initio unit cell determination and atomic-scale structure solution. The method is benchmarked using microcrystals of the small-molecule photoswitch sodium nitroprusside dihydrate, obtaining complete data to d_min = 0.6 Å by combining just 66 partial datasets selected against rigorous quality criteria.

Fig. 1. Crystal loading into the fixed target serial grid.

a Schematic description of the components in a fixed-target grid, and images of a fixed-target grid taken by scanning electron microscopy (SEM) to show: b the entire grid (×35 zoom), c a section of wells containing a single crystal (×270 zoom), and d a single well occupied by two crystals (×600 zoom). The grids were loaded using a 2.5 µL aliquot of a [0.08 g/g] suspension of crystals in 1-butanol.

Scheme 1

SR-FT-SSX Workflow. Flow chart describing the SR-FT-SSX method workflow for the study of known and unknown crystal systems.

Fig. 2. Fixed-target grid mounting on the synchrotron beamline.

Images of the fixed-target serial grid mounted on a modified magnetic base and on the diffractometer in Experimental Hutch 1 on Beamline I19 at Diamond Light Source. a Serial chip viewed from below the sample position, b serial chip view from side-on, c illustration of the XY rastering and small angle rotation procedure used during the FT-SR-SSX data collection.

Fig. 3. SR-FT-SSX data processing.

Visual representations of the fixed-target grid well positions in real space mapped to their dataset quality factor values, i.e. selection criteria used to select the most successful partial datasets, in the iterative processing route used to generate structure 2: a $R_{\mathrm{pim}}$ (yellow = high (poor) value, orange = low (good) value, blue = no value/crystal), b % of spots indexed (yellow = low (poor) % value, orange = high (good) % value and blue = no value/crystal) and c I/$\sigma$(I) (yellow = low (poor) value, orange = high (good) value, blue = no value/crystal), alongside histogram representations of the structure quality factors, d $R_{\mathrm{pim}}$, e % of spots indexed and f I/$\sigma$(I).