J. Appl. Cryst.の発刊に際して

We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.

/pdf/high-speed-fixed-target-serial-virus-crystallography-3tfac4fc0c.pdf

High-speed fixed-target serial virus crystallography.

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

/pdf/low-dose-fixed-target-serial-synchrotron-crystallography-5dji5twlxd.pdf

Low-dose fixed-target serial synchrotron crystallography

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures.

/pdf/room-temperature-macromolecular-crystallography-using-a-19hxgalvvw.pdf

Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering

Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

/pdf/strategies-for-sample-delivery-for-femtosecond-11811iyqe1.pdf

Strategies for sample delivery for femtosecond crystallography

The design and implementation of a compact and portable sample alignment system suitable for use at both synchrotron and free-electron laser (FEL) sources and its performance are described. The system provides the ability to quickly and reliably deliver large numbers of samples using the minimum amount of sample possible, through positioning of fixed target arrays into the X-ray beam. The combination of high-precision stages, high-quality sample viewing, a fast controller and a software layer overcome many of the challenges associated with sample alignment. A straightforward interface that minimizes setup and sample changeover time as well as simplifying communication with the stages during the experiment is also described, together with an intuitive naming convention for defining, tracking and locating sample positions. The setup allows the precise delivery of samples in predefined locations to a specific position in space and time, reliably and simply.

/pdf/a-modular-and-compact-portable-mini-endstation-for-high-112jptobiw.pdf

James O'Hea

Papers

A modular and compact portable mini-endstation for high-precision, high-speed fixed target serial crystallography at FEL and synchrotron sources