Proteins form the machines that carry out the essential functions of life. Like DNA and RNA, proteins are synthesized like "beads on a string" but with 20 different kinds of beads (amino acids) rather than the 4 of RNA or DNA. Chemical properties that distinguish different amino acids ultimately cause the protein chains to fold up into specific three-dimensional structures. It is the proteins that meet the third and greatest information challenge — which is to act out the instructions encoded in DNA. Although DNA and RNA are information rich, they are chemically simple and homogeneous. Proteins, by contrast, are chemically complex and diverse, properties that enable them to do so many different jobs. Proteins are "where the action is" in living systems. They are motors, pumps, chemical catalysts, detectors, signals and signalers, conveyers, structural units, gateway keepers, dismantlers, assemblers, and garbage handlers. They regulate cell replication, survival, and even death. Recent progress in whole-genome DNA sequencing and in areas of protein-structure determination have brought investigators to the point of knowing the composition of most proteins from model organisms, but the challenge is to know how proteins give cells their capabilities, structure, and higher-order properties.

From Genes to Life: a Primer

To understand how a protein does its job, it is not enough to know simply the kind and number of atoms that make it up; it is necessary to know the structure of the protein – the detailed arrangement of its atoms. Protein crystallography using storage ring synchrotron light sources has made it possible to determine the structures of many proteins. However even the brightest storage ring x-ray sources require that the protein be prepared as a crystal – an orderly array of protein molecules, spaced and oriented identically. The millions of molecules in the crystal scatter x-rays in a distinctive pattern that can be used to determine the structure of the protein molecule. The necessary scattering data can be collected in only a few hours at a synchrotron. The difficult part of protein crystallography is producing a usable crystal – this takes 99% of the time and effort. Indeed, some of the proteins most important to life processes are difficult or impossible to crystallize. If it were possible to determine the structure of a protein without the need to form a crystal, progress in understanding proteins could be accelerated one hundredfold.

The high intensity and short pulse duration of the LCLS beam could dramatically speed the determination of protein structures. The LCLS is so intense that smaller samples of proteins (perhaps a few hundred molecules) might be large enough to yield the necessary scattering data. High intensity poses a challenge as well- the proteins are damaged by the x-rays, distorting or destroying their structure. The very short pulses of the LCLS may provide a solution to the damage problem. It takes a very short time for the structure of a damaged protein to change- a time comparable to the pulse duration of the LCLS. Computer simulations show that it might be possible for a sufficiently intense and short x-ray pulse to scatter from a molecule or molecular cluster, producing a good scattering pattern in a time so short that the atoms in the damaged molecules have had no time to move.

At the outer extremes of these limits (not reached by LCLS in the first phase of its operation), scattering to high resolution may be recorded from large single macromolecular structures, viruses, nanocrystals, and nanoclusters of proteins without the need to amplify scattered radiation through Bragg reflections. Nevertheless, very important new experiments can be performed initially with the LCLS, even as an ongoing machine R&D program progresses toward approaching these limits. Averaging procedures can be applied to extend resolution when a reproducible sample scatters a sufficiently large number of photons for its orientation to be determined. Large samples scatter more x-rays (even if they have no internal symmetry), and thus their orientation is easier to determine than the orientation of smaller molecules. In the first instance, nanocrystals, nanoclusters of proteins, and individual virus particles will be studied. Holographic imaging, the utilization of increased radiation tolerance in short and intense x-ray pulses, numerical alignment and averaging of many images will give increased resolution on such large and reproducible macromolecular structures. The planned studies with the LCLS will explore these extraordinary possibilities. The short time structure of the source will allow a range of novel time-dependent experiments in which femtosecond spectroscopy can be combined with very fast structural studies (cf. proposal on femtochemistry). While certain key reactions in life are photochemical, most enzymes participate in diffusion-dominated processes with their reactants and partners. Time-resolved structural studies on diffusive processes in crystalline enzymes are difficult due to problems with mixing enzyme and reactant in the crystal. With submicron-sized samples, the vast majority of solution techniques and methodologies will suddenly become available for time-resolved structural investigations at the LCLS. X-ray diffraction tomography will be performed with the unfocused LCLS beam on whole cells at “intermediate” resolutions. With nonreproducible structures (e.g., living cells) or with reproducible but small structures (e.g., single protein molecules), higher resolutions could only be reached with a focused beam and with shorter pulses than the pulses planned initially at the LCLS. Thus, ultra-short and high-intensity x-ray pulses from the LCLS, in combination with novel container-free nanoscale sample handling methods, will open up amazing new possibilities for structural determinations with x-rays and may lead eventually to high-resolution experiments on nonrepetitive and nonreproducible structures like cells. This is a “never seen” regime where only predictions and simulations exist today.