Molecular structures may soon be exposed with the help of extremely bright ultrafast x-ray pulses tightly focused to cross a “particle beam” injector that fires micron-size droplets of water containing a single protein, virus or nanocrystal. The technique is so fast that it can capture diffraction patterns of atoms and biomolecules in motion. Combining these devices has heightened drug developers’ understanding of protein-drug bonding and cellular interactions for therapeutic drug development.Too big or too smallX-ray crystallography, the current method for protein structure analysis, uses a crystal comprising copies of proteins or viruses. A crystallized sample rotates under an x-ray beam, and the molecule scatters to reveal complex diffracting patterns that are converted into a three-dimensional image, displaying how the electrons and atoms are arranged. However, the visualization process can be difficult because more complex molecules, such as multiprotein assemblies and membrane proteins – important for the delivery of drug molecules through the cell membrane – may be too large to crystallize.By providing an aqueous environment for proteins, the molecules retain their biological atomic structure, essential for observing their natural state and functionality.“The aim of our project is to determine the atomic structure of proteins and viruses which cannot be crystallized or those which only form very small [submicron] crystals, not large enough for conventional crystallography,” said John C.H. Spence, co-designer of the “particle beam” injector and a physicist at Arizona State University.Just rightThe first components of the technology are the ultrabright, ultrafast x-ray pulses designed by researchers at Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory in Menlo Park, Calif. Their device, called a Linac Coherent Light Source (LCLS), is a free-electron laser that emits 100-fs pulses at a rate of 50 per second with an energy level between 2 and 8 kV. The pulses are more than a billion times brighter than modern electron accelerators. “These pulses are so short, they terminate before atoms get a chance to move,” Spence said.The fast pulse also helps eliminate the risk of radiation damage to a molecule, which can occur because of long exposure; however, the atom absorbs so much energy that it later explodes, resulting in a “diffract and destroy” method, Spence said.The second component, created by Spence, professor Bruce Doak and their colleagues at the university, is a gas dynamic virtual nozzle that helps maintain a protein’s precise atomic structure by hydrating the molecule and providing a natural aqueous environment.“The liquid drops encapsulate small samples of interest to biology, such as cells, microcrystals or individual proteins,” said Daniel DePonte, a postdoctoral student at Arizona State who refined the nozzle with physics research professor Uwe Weierstall. “The x-ray pulses serve to probe contents of the drops,” DePonte explained.Via a piezo vibrator, droplets are controlled to cross paths simultaneously with the x-ray pulses. The pulses are so fast that they freeze a protein’s orientation to capture 2-D diffraction patterns before the molecule can move or experience radiation damage effects caused by the beam’s energy. Diffraction patterns are constructed by revealing various angles of several biomolecules. Overlapping views are necessary to generate 3-D images of the structures.The micron-size droplets are fired through the nozzle at a flow rate of >100,000 times a second. DePonte said that helium was used for the acceleration of the droplets. “Rather than using gravitational force, the jets we use are generated using the gas dynamic forces of a co-flowing, coaxial, helium gas sheath, which provides a million times greater acceleration than that due to gravity.”Freezing timeDiffraction patterns of a molecular structure are generated by two-dimensional projections, Spence said. “The molecules come along in random orientation, and we are reading out about fifty of these 2k × 2k CCD diffraction patterns per second – a huge amount of data.” For overlapping views required for a 3-D image of the structure, each pattern originates from an identical biomolecule at different angles.The particle gun, combined with coherent x-ray technology, was successfully tested all last year at the Advanced Light Source, a division of Lawrence Berkeley National Laboratory in Berkeley, Calif. In December, another set of trials on the device will begin. The x-ray beam will be conducted at 2 keV, with a pulsed beam for low-resolution snapshots of molecular envelopes. The LCLS also is projected to operate at 8 keV by 2010 to reveal secondary protein structures.