Novel X-ray technique opens door to new biological insights

First high-resolution protein structure analysis demonstrates potential of free-electron lasers

Diffraction pattern of a lysozyme micro crystal illuminated by an X-ray laser flash. Image: Anton Barty/DESY

Hamburg, 31 May 2012. Scientists have used a novel X-ray technique to peer into the internal structure of a common biomolecule. The study, published in the journal "Science", demonstrates the immense potential of new tools called free-electron lasers (FEL) to obtain high-resolution structural insight into macromolecules. With the Linac Coherent Light Source (LCLS) at the U.S. SLAC National Accelerator Laboratory in California, the international team, including researchers from the Center for Free-Electron Laser Science (CFEL) at the campus of the German accelerator centre DESY in Hamburg, determined the structure of hen egg white lysozyme down to a resolution of 0.19 nanometres. Lysozyme was chosen as an already well-characterized model system to provide a proof-of-principle for the new technique. Despite the blazing intensity of the FEL X-ray pulses, which completely destroy the sample upon exposure, the method delivers high quality structural data, reports the team headed by SLAC scientist Sébastien Boutet.

“The exceptionally intense X-ray pulses possible with FELs open the door for analyzing completely new classes of biomolecules like proteins from the cell membrane, that are hard or nearly impossible to crystallize”, explains co-author and CFEL scientist Henry Chapman of DESY: “This will allow us to explore uncharted terrain in structural biology.” CFEL is a joint venture of DESY, the Max Planck Society and the University of Hamburg.

The structure of biomolecules is of great interest for medicine and biology, because their shape often determines their function. For example, when the structure of an enzyme critical to a cell receptor is known, it may be possible to design tailor-made medication. These structures are commonly determined by X-ray crystallography, in which crystallized samples are illuminated by an X-ray beam. The X-ray light is scattered by the regular arrangement of molecules in the crystal lattice and the resulting diffraction pattern allows the molecular structure to be calculated. However, the crystallization of many biomolecules is difficult and slow – with a large failure rate.

Lysozyme is one of the best-characterized biomolecules, and its structure has been thoroughly investigated at conventional X-ray sources. For precisely this reason the team chose this protein as a known model system to demonstrate structural determination using an FEL to a resolution sufficiently high to see individual amino acids. The structure obtained from the FEL data matches well the known structure of lysozyme, obtained separately using much larger crystals. “The good agreement benchmarks the method, making it a valuable tool for systems that yield only tiny crystals,” says co-author Ilme Schlichting from the Max Planck Institute for Medical Research in Heidelberg.

Novel X-ray lasers, such as the LCLS used in this study or the European XFEL currently being built in Hamburg, offer the possibility to study previously intractable molecular structures. The X-ray flashes from X-ray lasers are extremely bright so that only tiniest crystals are needed for a structure analysis. In fact, the micro crystals used in the study, measuring typically only 1 x 1 x 3 micrometres, almost immediately vaporize when subjected to the intense X-ray light. A flash from the LCLS is so short (only 5 million billionth of a second, or 5 femtoseconds) that it passes through the sample, carrying with it the information to form the diffraction pattern, all before the crystal has started to explode. “We were able to show that atomic resolution information can be collected before radiation damage has a chance to take effect,” explains co-author and DESY scientist Anton Barty from the Chapman group. “The key is ultrashort pulses – we see no effects of damage before the X-ray pulse has already passed.”

The team previously demonstrated this new technique using different biomolecules. Prior to the published work in “Science”, the obtained resolution was limited, however thanks to a new instrument at LCLS, the Boutet research team and collaborators were able for the first time to reach the resolution needed for detailed structural insight. The Coherent X-ray Imaging instrument (CXI) uses pulses of X-ray radiation with a wavelength of 0.132 nanometres generated by the LCLS (one nanometre is a millionth of a millimetre; visible light has wavelength between 400 and 800 nanometres).

Millions of small lysozyme crystals flowed through the laser beam in a liquid jet. From approximately 3.5 million X-ray flashes, the scientists registered about 100 000 hits, enabling the lysozyme structure to be determined with a resolution of 0.19 nanometres. “We’re able to use the powerful pulses from LCLS to actually visualize the structure of the molecule at a resolution so high we start to infer the position of individual atoms,” says Boutet. “This important demonstration shows that the technique works and it paves the way for a lot of exciting experiments to come.”

About CFEL
The Center for Free-Electron Laser Science (CFEL) on the DESY campus Hamburg-Bahrenfeld is a joint enterprise of Deutsches Elektronen-Synchrotron DESY, the Max Planck Society and the University of Hamburg. CFEL is designed to advance science with so-called free-electron lasers (FELs). These novel light sources based on linear particle accelerators enable to observe nature live on the scale of single molecules and atoms. Leading scientists of various disciplines meet under the CFEL roof to work jointly on interdisciplinary themes. Currently, more than 140 CFEL colleagues form five divisions and two Advanced Study Groups.

About LCLS
The Linac Coherent Light Source is a U.S. Department of Energy Office of Science-funded facility located at the SLAC National Accelerator Laboratory. LCLS is the world's first hard X-ray free-electron laser, allowing researchers to see atomic-scale detail on ultrafast timescales. The LCLS enables groundbreaking research in physics, chemistry, structural biology, energy science and many other diverse fields.

Original publication
"High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography"; Sébastien Boutet et al.; "Science", DOI: 10.1126/science.1217737

Science contact
Prof. Henry Chapman, Centre for Free-Electron Laser Science (CFEL), Tel. +49 40 8998-4155,
Dr. Anton Barty, Centre for Free-Electron Laser Science (CFEL), Tel. +49 40 8998-5783,

Press contact:
DESY Press Officer Dr. Thomas Zoufal, Tel. +49 40 8998-1666, <>



Experimental set up at LCLS. Image: SLAC National Accelerator Center

A) Electron density map of lysozyme at 0.19 nanometres resolution. B) Difference to synchrotron structural data for lysozyme. No interpretable features (red and green) are apparent. Image: Thomas R. M. Barends/Max-Planck-Institut für Medizinische Forschung

Coloured diffraction image of lysozyme in the X-ray laser beam. Image: Anton Barty/DESY

Coloured diffraction image of a lysozyme micro crystal in the X-ray laser beam. Image: Anton Barty/DESY

Rendering of a lysozyme molecule against its diffraction pattern in an X-ray laser. Image: Anton Barty/DESY