ID14-2 Macromolecular Crystallography Beamline
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ID14-2 is now closed to users and activities moved to ID30 (MASSIF)
Synopsis
Station ID14-2 is one of the four macromolecular crystallography beamlines using the highly intense ID14 undulators. Full user mode commenced in September 1999. It is a fixed energy station dedicated to high throughput structural biology.
The beamline is designed to achieve the following:
- a monochromatic X-ray beam of fixed wavelength 0.933Å (13.294keV);
- a beamsize of 50 to 200 microns (using slits) and smaller possible (beam down to 10 microns has been used);
- a focal spot size of 400 microns horizontally and 200 microns vertically
- a maximum resolution of 0.97Å.
The beamline is equipped with a mini-diffractometer with motorised z-translation and x/y sample alignment translations and a miniKappa goniometer, ADSC Q4 CCD detector, an Oxford Cryostream for 100K data collection and an automated sample changer. All the ESRF macromolecular crystallography beamlines have a common data collection environment called mxCuBE. Data turnover of between 5 to 6 images per minute can be achieved based on 1s exposures and a 5s readout from the ADSC Q4 detector. Typical exposures are 1s to 10s per degree. The X-ray wavelength is below most interesting absorption edges, and this has been exploited in the use of SAD phasing to solve several structures. The beamline is also equipped with an XFlash fluorescence detector enabling X-ray fluorescence emission spectra (XFE) to be performed. This can identify elements, with an absorption edge below 13.294 keV, that may be bound to the protein. Other devices can be mounted on request such as the Humidity Control Device from the Diffraction Instrumentation Group at the EMBL, see here.
Scientific Applications
"The ultimate goal of molecular biology is to understand biological
processes in terms of the chemistry and physics of the macromolecules that participate
in them. One of the essential differences between the chemistry of living systems
and that of the non-living is the great structural complexity of biological
macromolecules. We shall not unravel the chemistry of life without knowing at
atomic or close to atomic resolution the structure of biological macromolecules,
especially the proteins."
Preface, Introduction to Protein Structure, C. Branden & John Tooze, Garland
Publishing Inc., 1991.
The biological function of a macromolecule is intimately related to its structure. In order to understand the function of a protein we therefore need to know its structure on an atomic scale. Diffraction from macromolecular crystals leads to the calculation of electron density maps. These are then used to the construct models of the distribution in real space of atoms in the macromolecule.
The advent of recombinant gene technology and protein expression systems in combination with the publication of genomes for many organisms, including man and many of his pathogens, has led to a surge in the number of available macromolecular crystals from which data needs to be collected. There is great interest within the scientific community to understand the function of the expression products of these genomes and an aid to the understanding of biological function is knowledge of macromolecular structure. X-ray crystallography is by far the dominant technique used in solving such structures and the advent of third generation synchrotron sources, such as the ESRF, is proving a great benefit to the rapid collection of X-ray data from crystals of biological interest.
Macromolecules of interest to the scientific community are many and varied. They range from apparently simple monomeric proteins (e.g. haemoglobin), to multimeric complexes (e.g. the ribosome) and complete viruses. Researchers from throughout Europe and beyond are studying biological processes as diverse as DNA recombination and repair, metabolism, cell cycle control, transcription, transport, etc, all with the aim of relating function and structure.
ID14-2 was designed to be simple to operate and projects are constantly undergoing within the MX group to increase the automation of the beamline control and data collection and processing. In this way ID14-2 will continue to be used for very rapid collection of data from macromolecular crystals.
Techniques Available
Fixed wavelength rapid data collection and SAD/MIR.
Useful links
- ISPyB
- International Tables A
- Why you don't need to worry too much about the crystals in your dewar
- DNA manual
- Sample changer manual
- Setting up your laptop on the beamline
- Users' safety training
- mosflm manual
- ccp4 documentation
- coot manual