Schematic representation of the experimental setup at the Coherent X-ray Imaging endstation at the Linac Coherent Light Source. Millions of tiny crystals are injected into the free-electron laser beam in a thin liquid jet. Diffraction patterns are generated when a crystal intersects a free-electron X-ray flash and are captured on a detector shown on the left. Image: MPI for Medical Research

In
the centennial year of Max von Laue’s discovery that X-ray diffraction
can be used to unravel the atomic architecture of molecules, a new
approach to the determination of high-resolution structures has been
demonstrated. An international team of researchers has analyzed tiny
protein crystals using short pulses of X-ray light from the world’s
first hard X-ray free-electron laser, the U.S. Department of Energy’s $300
million Linac Coherent Light Source at Stanford.

The
study demonstrates the immense potential of free-electron lasers for
obtaining the structures of macromolecules from tiny crystals when
illuminated with the blazing intensity of the ultrashort free-electron
laser X-ray pulses, even though the crystals are destroyed in the
process. In the current study, their structural analysis reveals details
with a spatial resolution of 0.2 millionth of a millimeter. The team,
including researchers from the Max Planck Institute for Medical Research
in Heidelberg and the Max Planck Advanced Study Group in Hamburg,
showed that their data compared well with those collected from large,
well-characterized crystals using conventional X-ray sources, providing a
benchmark for the new free-electron laser approach. Their
proof-of-principle experiment shows that the free-electron laser is an
important new tool for structural biology on large macromolecular
assemblies and membrane proteins, many of which are known to be
important targets for pharmaceutical development.

X-ray
free-electron lasers are extremely powerful new X-ray sources that
provide highly intense ultrashort flashes of light. The intensity of
such an X-ray pulse is more than a billion times higher than that
provided by the most brilliant state-of-the-art X-ray sources, with a
thousand-fold shorter pulse length, on the order of a few millionths of a
billionth of a second, or femtoseconds. These properties provide
scientists with novel tools to explore the nano-world, including the
structure of biological materials.

Most
of our knowledge of the three-dimensional spatial architecture of
molecules has been obtained by X-ray crystallography, which relies on
the amplification of the scattering signal of the molecules by their
arrangement into relatively large crystals, often on the order of some
tenths of a millimeter. Obtaining large crystals can be extremely
difficult in the case of bio-molecules due to their inherent instability
and flexibility, as well as their typical low abundance.

Structure of the protein lysozyme. The spatial arrangement of the 129 amino acids is schematically depicted in the form of spirals (helices) and arrows (pleated sheets). Image: MPI for Medical Research

Free-electron
lasers can obtain structural information from tiny crystals that refuse
to reveal their secrets by conventional structural methods due to the
damage induced by the radiation used for the structure analysis.
Although the tiny crystals are completely destroyed by the incredibly
intensity of the free-electron laser, the ultra-short pulses can pass
through the sample before the onset of detectable damage and thus
provide the necessary scattering signal of the still-intact molecules.

In
this diffraction-before-destruction approach, crystals are replenished
for serial data collection by injecting them into the free-electron
laser beam using a liquid jet, developed by scientists from Arizona
State University, exposing one crystal after the other instead of
rotating a single large crystal as in conventional crystallography. This
concept of serial femtosecond crystallography has been demonstrated
before by the same team of researchers at the Linac Coherent Light
Source at Stanford, using the CAMP instrument, developed by the Max
Planck Advanced Study Group. The relatively long wavelength X-rays then
available limited the attainable level of structural detail.

Very
recently, a new instrument at the Linac Coherent Light Source, the
Coherent X-ray Imaging endstation, has allowed the use of short
wavelength X-rays and thus made it possible to infer atomic detail in
the molecular architecture. To benchmark the method, a
well-characterized model system was investigated, the small protein
lysozyme, the first enzyme ever to have its structure revealed.

The
researchers collated ten thousand snapshot exposures from crystals that
measured only a thousandth of a millimeter, and showed that the data
compared well with those collected using conventional approaches and
hundred-fold larger lysozyme crystals. Importantly, no significant signs
of radiation damage were detected.

“This
proof-of-principle experiment shows that the X-ray free-electron laser
indeed lives up to its promise as an important new tool for structural
biology on large macromolecular assemblies and membrane proteins. It
really opens up a completely new terrain in structural biology”, Ilme
Schlichting, leading the Max-Planck team, says.

Since
small crystals are typically easier to produce than large ones, this is
of immediate relevance for all studies of molecules that are difficult
to crystallize—as are some 60% of all proteins, many of which are prime
targets for medical therapies.

Source: Max Planck Institute