The large multi-component complexes and membrane proteins now routinely studied in structural biology tend to produce either very small crystals or crystals that can be extremely heterogeneous in their diffraction properties. In order to locate very small crystals or the optimum region of a crystal larger than the X-ray beam, mesh scans have been developed to collect an image at numerous points specified within a grid. However, final centring can remain challenging. This workflow scans the face of a loop using a mesh scan and then performs a line scan 90 degrees away on the best area of diffraction determined from the first scan.  The best volume is then automatically centred to the beam, ready for final characterisation and data collection.

 

The process is fully described in this publication:

Svensson, O., Monaco, S., Popov, A. N., Nurizzo, D. & Bowler, M. W. (2015). Fully automatic characterization and data collection from crystals of biological macromolecules, Acta Cryst. D71, 1757-1767

Please cite if you found  X-ray centring useful in screening, data collection or structure solution.

 

 

 

Contact Matthew Bowler for help.

 

The workflow is started by selecting the "X-ray Centring" workflow in the Advanced tab and adding to the queue:

 

The subsequent option is to then define a mesh over the area of interest. This can be done in the usual manner in the "Advanced" tab using the "Grab" tool and specifying number of vertical and horizontal steps:

 

mxcube_WF_18.png

 

This step can be performed before or after starting the workflow. By default, all images have a "mesh-" prefix and are stored in a subdirectory of the current working directory called "mesh###". Images are collected at each of the intersecting points of the mesh with run number 1 for the first mesh and 2 for the second dimension 90 degrees away. The next step is to define exposure time and image prefix:

 

WF_new_13.png

 

After data collection has finished a map is produced in the directory showing the results of analysing the images:

WF_MESH_2.png

The workflow will then rotate the crystal 90 degrees away from the current position and move the crystal to the best position determined from the first orientation.  A line scan is then proposed - here the number of steps and distance can be altered from the default (the default is the distance determined from the shadow of the loop divided by the beamsize).  On clicking "continue" the collection is loaded to the queue. Again analysis is performed and a second map generated, the crystal is then automatically centred to the optimum diffraction volume.  You can now continue with data collection

WF_MESH_3.png

 

 

 

 

 

 

 diffraction_map0_a

 

diffraction_map90

 

 

The centring is extremely accurate, below is an example of locating and centring a 20 um needle to a 5 um beam (ID23-2).  After the workflow centring proceedure a full data set was collected and the structure solved. mesh_and_scans_p38

The workflow can also be used to locate and centre small crystals, in this case the best 10 um crystal was centred to a 10 um beam:

 

 insulin_micromesh