Room Temperature Serial Crystallography at Synchrotrons

Abstract

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.

ID30A-3 (MASSIF-3) – a beamline for macromolecular crystallography at the ESRF with a small intense beam

“…In addition to being an excellent vehicle for standard MX experiments based around ESRF-developed automation, this setup has already proven very useful in the application of new diffraction data collection methods [i.e. MeshAndCollect (Zander et al, 2015)] and room-temperature synchrotron serial crystallography (SSX) data collection using sample delivery systems such as the high-viscosity extruder (HVE) system developed by Botha et al (2015).…”

Current status and future opportunities for serial crystallography at MAX IV Laboratory

“…Another way to perform SSX experiments at BioMAX is to use an HVE injector ( Fig. 3) which already produces rewarding results at other synchrotrons (Nogly et al, 2015;Botha et al, 2015;Martin-Garcia et al, 2017). The HVE injector at BioMAX was designed and fabricated at Max research papers (a) HVE injector mounted and prepared for the experiment.…”

“…The internal size of the silica capillary varies from 50 mm to 100 mm in diameter, depending on the crystal size expected to be used. The sampleloading procedure uses Hamilton syringes with volumes of 100 ml or 250 ml, as described in the work by Botha et al (2015). At the end of the nozzle, helium gas at a maximum pressure of 50 psi flows through a borosilicate sheath capillary [ Fig.…”

Making a difference in multi-data-set crystallography: simple and deterministic data-scaling/selection methods

“…The term 'SSX' has recently been used in a wider sense, referring to fixed-target or injection-based single zero-rotation diffraction patterns (stills) from crystals exposed to monochromatic (Nogly et al, 2015;Botha et al, 2015;Owen et al, 2017) or polychromatic (pink) radiation (Meents et al, 2017;Martin-Garcia et al, 2019). Serial femtosecond crystallography (SFX) takes this method to the extreme; it collects stills from numerous small crystals before destroying them using X-ray pulses generated by a free-electron laser.…”