The future is bright-- structural biology at FELs

Protein crystallography using synchrotron radiation sources has had tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their working mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radiation damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures. With the advent of X-ray free-electron lasers (FELs) this situation appears remedied. Theoretical considerations had predicted that with sufficiently short pulses useful diffraction data can be collected before the onset of significant radiation damage that ultimately results in Coulomb explosion of the sample. This has been shown recently at the first hard X-ray FEL, the LCLS at Stanford. High resolution data collected of a stream of microcrystals of the model system lysozyme agree well with conventional data collected of a large macroscopic crystal [1] With the demonstration that de-novo phasing is feasible [2], serial femtosecond crystallography has been established as a useful tool for the analysis of tiny crystals [3] and thus the large group of proteins that resist yielding macroscopic crystals such as membrane proteins. In addition to ensure the required fast exchange of the microcrystals upon exposure, liquid jet delivery has the advantage of allowing data collection at room temperature. As demonstrated recently, this is important since structural dynamics and thus the observed conformation is often temperature dependent. Recent results will be described.

Room Temperature Serial Crystallography at Synchrotrons

Serial Femtosecond Crystallography (SFX) is the most commonly used method for the emerging structure determination at X-ray free-electron lasers (FELs). The high peak brilliance of the FEL and the possibility of using femtosecond pulses afford use of nano-to-micron sized crystals in a diffraction-before-destruction approach for the acquisition of high-resolution undamaged diffraction data [1]. The crystals are obliterated upon exposure to an FEL X-ray pulse so only a single snapshot can be collected per crystal, necessitating a constant supply of fresh crystals. The crystals are therefore injected in a liquid microjet [2], [3]. We show that this serial method of data collection and the associated data analysis can be successfully adapted to serial crystallography (SX) measurements at synchrotrons, enabling room temperature studies using the unattenuated beam. Given the continuous supply of fresh crystals, the full tolerable dose can be used for each single crystal exposure, permitting analysis of small or weakly scattering crystals. FEL X-ray pulses are much shorter than the fraction of a second exposure time at a synchrotron, so SFX injection conditions are modified in SX such as to slow down the typically fast travelling crystals. By embedding the crystals in a viscous material the crystals remain in the beam long enough to yield measurable diffraction and smearing out of the diffraction peaks due to crystal tumbling is avoided. We demonstrate the successful application of room temperature SX at the Swiss Light Source at ambient pressure. Our experimental setup allows collection of both still and rotation data. Recent progress using model systems will be presented, establishing this high throughput, high dose rate approach as a new route to structure determination of macromolecules in their native environment and at room temperature.

De novo phasing of protein crystallography data from a free-electron laser

Free-electron lasers (FELs) are pushing back the limits of possibility in protein crystallography. Using the high-intensity, femtosecond duration pulses afforded by FELs allow data collection from micrometer-sized crystals while outrunning radiation damage. Moreover, FELs may be used for pump-probe experiments with unprecedented time resolution. However, the intricacies of FEL data collection pose specific challenges: as every FEL pulse destroys the sample, data are mostly collected from a stream of microcrystals and averaged to remove the variations in crystal size and quality as well as shot-to-shot variations in beam parameters. This technique is called serial femtosecond crystallography (SFX). In SFX, several tens of thousands of images typically need to be averaged to obtain reasonably accurate structure factor amplitudes. We previously showed that SFX yields structure factor amplitudes accurate enough to detect the weak anomalous signal of endogenous sulfur atoms. Now we show that SFX can be used to collect data accurate enough for de-novo phasing of a protein structure[1]. Using a model system (gadolinium-derivatized lysozyme) we collected ~60,000 diffraction images and obtained structure factor amplitudes that allowed phasing by single-wavelength anomalous diffraction. This first demonstration of de novo phasing from FEL data leads us to anticipate that FEL-based crystallography will become an important tool for the structure determination of proteins that are extremely radiation sensitive or that are difficult to crystallize, such as membrane proteins.