to BioTechniques free email alert service to receive content updates.
Diffraction before Destruction | Microscopy Feature

Ansa Varughese

For years, researchers have struggled with crystallizing samples to determine the structure of biological molecules via X-ray diffraction. But a new breed of free-electron lasers that generate intense, pulsed light beams is changing that. Ansa Varughese reports. 

Bookmark and Share

In April 2009, researchers at the SLAC National Accelerator Laboratory in Menlo Park, CA, fired up the world’s brightest X-ray light source: the Linac Coherent Light Source (LCLS). The short X-ray laser pulses promised to provide a clearer picture of the 3-D atomic arrangements of various materials, including biological molecules.

The LCLS Atomic, Molecular, and Optical instrument hutch where Chapman and colleagues performed the first FEL experiments. Source: Brad Plummer, SLAS

"This milestone establishes proof-of-concept for this incredible machine, the first of its kind," said former SLAC director Persis Drell in a statement at the time. "The LCLS team overcame unprecedented technical challenges to make this happen, and their work will enable frontier research in a host of fields. For some disciplines, this tool will be as important to the future as the microscope has been to the past."

After more than two years of fine-tuning and experimentation, the LCLS team is now obtaining structural data from biomolecules that were previously inaccessible. While researchers using other light sources struggle to prepare protein crystals of sufficient size for structure determination, LCLS researchers can obtain data from relatively small crystals.

In addition, the LCLS is providing information about simple biological organisms, including viruses and microbes. And with this new data, scientists are developing new theories on evolution and function.

Waiting for the FEL

Henry Chapman’s patience has been tested over the past decade. That’s how long Chapman and colleagues at the Center for Free-Electron Laser Science at the Deutsches Elektronen Synchrotron (DESY) in Hamburg, Germany, have been discussing a way to improve the structure determination of biological molecules. By using an intense femtosecond X-ray pulse, they believed that a diffraction pattern could be recorded from a molecule before being destroyed by the radiation.

The only problem was producing such an intense X-ray beam. While third-generation synchrotron light sources have been excelling at obtaining diffraction patterns from rather large protein crystals, Chapman’s technique would require a much more powerful light source, namely, an X-ray free-electron laser (FEL).

But such an X-ray FEL didn’t exist. While organizations like the SLAC and the DESY proposed constructing a FEL during the 1990s, support was scarce because the scientific potential of these lasers had not been fully understood. At the time, these proposals were considered risky and expensive ventures, costing more than $1 billion to construct.

So, Chapman and his colleagues had to wait, carrying out computer simulations of the technique to show the potential of structural determination of biological molecules using a FEL (1). They believed that FELs could allow researchers to obtain diffraction patterns not only from crystallized protein but also from non-crystallized protein, other biological molecules, and even cells.

In 2006, with the support of the US Department of Energy, the SLAC National Accelerator Laboratory finally began construction of the LCLS to house the world’s first FEL. In order to produce such an intense light beam, the electron beam had to be constructed with extreme precision, deviating less than 5 mm per 5 meters.

Three years later, the SLAC researchers fired up the laser for the first time, producing the world’s brightest light beam ever. In their initial tests, the researchers produced a laser light with a wavelength of 1.5 Angstroms—the shortest-wavelength, highest-energy X-rays ever created.

After several months of fine-tuning the FEL, the LCLS was officially open to collaborators for experimentation. And Chapman was one of the first in line.

First Results

Researchers John Spence, Henry Chapman, Inger Andersson, and Janos Hajdu at the LCLS. Source: R. Brude Doak, Arizona State University

By mid-2010, Chapman had already analyzed the results of his experiments at the LCLS. Everything that he believed about the potential of FELs turned out to be true. The short and intense pulses produced diffraction patterns of biological molecules before the sample was destroyed.

One of the first tests that Chapman and colleagues decided to initiate was to obtain diffraction patterns of proteins that were difficult to crystallize. Previously, researchers have used X-ray crystallography to determine the structure of proteins.

To obtain good diffraction patterns using a traditional X-ray source, protein crystals need to be of substantial size. Crystals as large as one millimeter were needed for standard X-ray crystallography. Although the light source’s intensity can be increased to obtain diffraction patterns from smaller crystals, the radiation destroys the sample before these patterns can be recorded.

As a result, researchers have struggled to determine the structure of proteins that do not crystallize easily, such as membrane proteins. But these difficult-to-crystallize proteins have important biological functions and could be potential targets for new drugs and treatments. So, Chapman and colleagues wanted to see if they could determine the structure of proteins that only produced nanometer-sized crystals by pulsing the intense light beam from the FEL.

To deliver nanometer-sized crystals to the pulsed X-ray beam, the group turned to Arizona State University physicist John Spence who designed a special liquid jet. The jet has a gas flow around it that speeds up the liquid holding the crystals and then focuses the liquid through the small reaction chamber. The crystals flow at 10 m/s and each pulse repeats at a rate of 30Hz, resulting in 1800 recorded diffraction patterns per minute.

“This way of using a really short pulse allows you to put the required amount of X-rays on to the samples and get the diffraction pattern, and all that happens before the object knows what’s hit it,” says Chapman.

As a result, they obtained millions of diffraction patterns that they could then assemble into a 3-D structure. With the FEL, the team recorded diffraction patterns from samples as small as 100 nanometers. This also makes it easier to grow crystals, because they’re much smaller. In a paper published in Nature in February 2011, Chapman and colleagues presented their method for structure determination of nanocrystals using femtosecond pulses of intense X-ray light from the LCLS (2).

Because the pulses are so fast, the researchers obtain gigabytes of data per minute and terabytes of data per experiment—leading to more data, maybe too much, to sort through.

“We don’t need to measure it with so much data as we do now,” says Chapman . “We need to be able to get good structural information with fewer data.”

Also, the flow from Spence’s jet is continuous, wasting a lot of crystallized proteins to get the data. In the future, Chapman hopes that they can develop a pulsing delivery system that expands upon the current jet to minimize the number of crystals required for structure determination.

Beyond proteins

X-ray diffraction pattern of a single mimivirus particle. Source: Tomas Ekeberg, Uppsala University

In addition to protein determination, Chapman and colleagues designed another experiment that would allow them to study other biological molecules at the atomic level. But this time, the samples required no crystallization.

In a paper published in Nature in Febraury 2011, the team reported the structure of a single mimivirus particle using these pulsed intense X-rays from the LCLS (2). Because the femtosecond pulse was so short, the particle suffered no measurable damage from the radiation exposure. In the end, after injecting hundreds of particles to the pulsing X-ray beam, two samples provided the team with enough data to reconstruct the structure.

But such experiments are just the beginning. Chapman believes that with shorter pulses and higher intensity, FELs could provide a new era of high-resolution imaging of biological samples, from viruses to singles cells and more.

Without the need for crystallization, Chapman believes that FELs will provide researchers with a whole new approach to understanding biology. For example, if one could synchronize the x-ray pulse with a specific trigger, they could collect data on chemical reactions in progress.

“In some way, you want get a time-resolved measurement—you want to see the reactions in progress, and the short X-ray pulse will give you a way to get a fast shutter speed [you need to capture these reactions in progress],” says Chapman.

By doing this, researchers can remove the delay between the trigger and the pulse, allowing them to capture a movie. They could also look at reactions by mixing substrates and crystals prior to being hit by the X-ray.

But the LCLS is the only FEL in the world, accommodating only one experiment at a time. So the surge of proposals has overwhelmed the facility, which has to choose the most interesting and promising ones from the masses.

Now, structural biologists are in the midst of a new period of waiting as other countries and organizations are now moving forward with their proposals to create their own FEL facilities after seeing the potential demonstrated by the LCLS facility. This will allow more researchers to experiment with the possibilities that these new breed of lasers have to offer. In 2013, the Center for Free-Electron Science in Hamburg, Germany hopes to have its FEL operational, which would allow Chapman to run his own experiments a little closer to home.


1. Miao, J, H.N. Chapman, J. Kirz, D. Sayre, and K.O. Hodgson. 2004. Taking X-ray diffraction to the limit: macromolecular structures from femtosecond X-ray pulses and diffraction microscopy of cells with synchrotron radiation. Annu Rev Biophys Biomol Struct. 33:157-76.

2. Chapman, H.N., P. Fromme, A. Barty, T.A. White, R.A. Kirian, A. Aquila, M.S. Hunter, J. Schulz, D.P. DePone, et al. 2011. Femtosecond X-ray protein nanocrystallography. Nature 470: 73-77.

3. Seibert, M.M., T. Ekeberg, F.R. Maia, M. Svenda, J. Andreasson, O. Jönsson, D. Odic, B. Iwan, A. Rocker, D. Westphal, et al. 2011. Single mimivirus particles intercepted and imaged with an X-ray laser. Nature 470: 78-81.