Fixed Target Serial Data Collection at Diamond Light Source

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

Serial synchrotron crystallography, or SSX, is a relatively new and rapidly developing area of MX.It's very well suited to low dose and room temperature experiments, but perhaps most importantly, it's really well suited to following dynamics. This allows stop-motion movies of proteins in action to be made. At I24, multiple modes of SSX are available.

This protocol focuses on fixed-target SSX, which allows a wide range of serial experiments using limited amounts of sample. The silicon chips can be damaged when loading and cleaning them, if you're unfamiliar with them. Also, the chip alignment protocol is completely new to many crystallographers, and the data processing is slightly different to your standard rotation data.

To prepare a chip holder, cut two sheets of polyester foil into approximately 6-by-6 centimeter squares, and place the sheets over the two base plates. Using metal sealing rings, fix the sheets in place, then carefully pull on the excess foil to remove any creases. Next, select a silicon chip with appropriately sized apertures relative to the size of the crystals to be analyzed.

Glow discharge the chip for 25 seconds at 0.39 millibar, with a 15 milliampere current. Use tweezers to place the silicon chip on the chip loading stage, with the raised bars facing down. Then, using a pipette, apply 200 microliters of the microcrystal slurry to the flat side of the chip, spreading the slurry to cover all the city blocks of the chip.

If the chip is damaged, cover any holes with a small piece of polyester foil, to ensure that an even vacuum can be applied. Then apply a gentle vacuum to aspirate all of the excess liquid through the chip. Remove the chip from the chip loading stage, and carefully blot the underside of the chip with filter paper to remove excess liquid.

Place the loaded chip on the larger half of the chip holder between the guide marks, flat side down. Place the small half of the chip holder onto the chip to seal it. The two halves of the chip holder should snap into place.

Then, using hex bolts, fix the chip securely in place. To align the chip, hold the chip holder at a 30-degree angle while approaching the mount. Then use the kinematic mounts to place the loaded chip onto the XYZ stage at the beam line.

When the magnets make contact, allow the chip holder to rotate parallel to the flow, and click into place. Once the chip is placed, use the beamline on-axis viewing system and the chip-alignment graphical user interface to locate the top left fiducial of the chip. To center on fiducial 0 in X, Y, and Z, move in and out of focus to align Z, up and down to align Y, and left and right to align X.Then click Set Fiducial 0"After aligning fiducial 1 and 2 with the X-ray beam in the same manner, click Make co-ordinate system"to generate a co-ordinate matrix.

Then click Block Check"to move the XYZ stage to the first well of each city block. If the X-ray crosshair lines up with the apertures, the chip is aligned. In this graphical representation of spot finding results from DIALS an updating hit-rate plot can be observed.

If a hit is clicked, the corresponding diffraction image will be displayed in the DIALS image viewer. In this table, current indexing and integration rates that update in real time as the data is collected during the visit can be visualized. Visualization of the unit cell parameters can reveal polymorphs.

Two-dimensional plots of useful parameters can also be produced to reveal variations that arise due to loading or dehydration effects. Stereographic projections can reveal the presence or absence of preferred orientations that can be fed back into the loading protocol. For example, in this projection, the effects of overloading a chip with lysozyme crystals can be observed.

When loading the chip, pipette slowly and use your finger to rest the pipette above the chip. This prevents you from touching the chip and accidentally stabbing holes in the silicon. When processing data, make use of the automated pipelines available.

This will give you a good idea as to how your experiment is progressing, and if you need to vary something. This method can be applied to any protein system that can be crystallized in significant quantities. And the reaction can be triggered by light X-rays or rapid mixing.

Dynamic studies on a series of structures can provide insight into how proteins function.