X-ray crystallography beamline

3D images of molecules at atomic scale for insights into molecular interactions and biological processes

 

Instruct has 3 centres offering X-ray crystallography beamline across Europe. Navigate the map and click on the pins to discover centres near you.

X-ray crystallography beamline Details

User Guide

Most synchrotrons already offer access to their suite of X-ray crystallography beamlines to applicants selected on the basis of peer-review. Applications for beamline time are generally made through web-based proposal systems and should be consulted first. Instruct hopes to provide an additional portal of access to some of these X-ray beamlines in the near future, once its application process has been implemented.

A typical X-ray diffraction experiment may take anything from a few (1-2 minutes) on an insertion device beamline, to tens of minutes on bending magnet beamlines. However, while the experiment itself is relatively fast, a number of crystals need to be screened for their diffraction properties, in order to select the optimal one or two for a successful experiment. For the most challenging structures, hundreds or even thousands of crystals may need to be screened.

This screening is normally facilitated by robotic sample changers, allowing many crystals to be tested. Users should mount their crystals onto SPINE standard pins and vial, which are supported by all European synchrotrons.

Most sites also provide online sample characterisation using programs such as EDNA (http://wiki.edna-site.org/index.php/Main_Page) and we recommend using such software for selecting the best diffracting crystals. These selection results are fed into databases such as ISPyB for further analysis and book-keeping. Once the best crystal has been remounted, data is carefully collected using an optimized strategy, again using EDNA.

Many sites now also offer automatic data processing, and indeed other automated structure solving routines, whose results are also saved in ISPyB. The last step is to ensure that data is properly backed-up, either using ftp servers or external hard drives. Please consult the beamline webpages before coming, to ensure you are able to retrieve or back-up your data.

Technical Specifications

An X-ray diffraction experiment is quite simple and only requires the accurate measurement of data. However macromolecule crystals are difficult to produce and can significantly vary in size and quality. Most modern synchrotron based X-ray crystallography beamlines are therefore designed to accommodate such challenges and typically contain the following major elements: A monochromator to select the desired wavelength (for phasing experiments); A focusing element to produce a small highly intense beam at the sample measurement position; A goniometer to accurately position crystals in such small X-ray beams; A fast and accurate X-ray detector; A high throughput sample changer to screen and select the best crystals for an experiment.

Such diverse equipment requires the development of sophisticated software algorithms to ensure movement is coordinated. Many of these routines are hidden behind easy and intuitive graphic user interfaces. The robustness of such software now allows remote data collection, saving users time and travel expenses.

Other software developments include the automatic characterization of samples to help select the best crystal, as well as processing the data as it is collected, which ensures that optimal data is collected before the sample is removed. Lastly, some more specialized X-ray crystallography beamlines allow access to ancillary equipment such as kappa goniometers for crystal reorientation, crystallization plate screening capabilities, dehydration devices and on-line microspectrophotometers.

  1. A monochromator
  2. Synchrotron radiation produces a spectrum of X-ray radiation, but most X-ray diffraction experiments require monochromatic X-rays. Monochromation is achieved by using a large crystal (silicon or diamond) oriented to diffract a certain wavelength. This can also be changed by rotating the diffraction plane.
  3. A Focusing element
  4. A synchrotron has a typical source size in the 1-2 mm range, while macromolecular crystals are typically < 100 um. It is therefore desirable to have an X-ray beam in the latter range to maximize the diffraction possible from such samples. The provision of smaller beam sizes (5-50 um) at the sample is achieved by using focusing elements. The beamline design generally dictates what focusing elements can be used. These vary from simple toroidal mirrors to more complex bimorph and Kirkpatrick-Baez focusing mirror, or newer technologies such as compound refractive lenses.
  5. A high precision gomiometer
  6. X-ray diffraction occurs in three dimensions and essentially as a sphere. However, X-ray detector are unable to collect data in a spherical geometry. Therefore, in order to collect all the diffraction data needed for a structure determination requires the accurate rotation of the crystals during a diffraction measurement, often synchronized with a millisecond shutter to allow for detector integration. All these frames are then merged for a complete dataset.
  7. A high throughput sample changer
  8. Macromolecular crystals are generally fragile and require extensive manual manipulation before they can be mounted at the sample position on an X-ray crystallography beamline. Manipulations include fishing the crystals from crystallization drops and flash freezing them in cryogenic conditions to try and mitigate against radiation damage. This results in macromolecular samples of variable quality. A number of crystals are therefore screened and the best one is chosen for optimal diffraction measurements. For more challenging projects large numbers of crystals need to be screened. Such large screening requires the extensive use of sample changing robotics and therefore most modern X-ray crystallography beamlines are equipped with such devices.