Published: February 26th, 2021
We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.
Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.
Serial synchrotron crystallography (SSX) is an emerging method of data collection which was inspired by X-ray free electron lasers (XFEL)1,2,3. At an XFEL, a single diffraction pattern is recorded from a usually very small protein crystal, before the crystal is destroyed by the extremely bright X-ray pulse. This means, typically, that a new crystal must be introduced into the X-ray beam to obtain another diffraction pattern4. This need to continually replenish crystals has driven the development of many serial sample delivery techniques5.
At synchrotrons, classic (non-serial) rotation crystallography methods are widely applied, exploiting a single large crystal which is rotated in an X-ray beam using a goniometer to collect a complete dataset for structure solution6. In order to increase the lifetime of crystals so that a complete dataset can be collected7,8, and also to facilitate shipping and automated sample transfer, crystals are cryocooled to ~100 K for data collection. At intense microfocus beamlines, multi-crystal strategies are frequently employed as radiation damage can prohibit the collection of a complete dataset from a single crystal9,10,11. Despite the limits imposed by radiation damage, the number of crystals used remains relatively modest and the approach used is essentially identical to the single crystal experiment.
SSX, on the other hand, uses serial sample delivery to obtain single still diffraction patterns from thousands of randomly orientated crystals to generate a complete dataset. It is noted that serial techniques incorporating crystal rotation are under development12,13 though we focus on still, zero rotation, approaches. There are a wide variety of sample delivery systems with different advantages and disadvantages14, ranging from delivering a stream of crystals in a flow focused/viscous jet15,16,17, microfluidic chip18,19, or crystals on a fixed target such as an etched silicon chip20,21. Typically, crystals are held at room temperature, allowing greater conformational diversity to be observed and providing a more physiologically relevant environment22. SSX enables the collection of very low dose datasets23, as the total dose of the dataset is equivalent to a single short X-ray exposure of one crystal. Another major advantage SSX provides is the study of protein dynamics through time-resolved methods, with reactions triggered by exposure to laser light24,25,26,27or by mixing of crystals and ligand/substrate28,29. Using smaller crystals means laser light can penetrate the entirety of the crystal, uniformly initiating the reaction without multiphoton absorption to provide well defined reaction intermediates for diffraction data taken at different time points27. Use of larger crystals and rotation-based data collection methods suffers from a limited laser penetration depth, nonuniform or multiphoton activation, radiation damage, and mechanical overhead time within data sweeps, resulting in a mix of reaction intermediates that can prove difficult or impossible to interpret at faster reaction speeds. Smaller crystals provide a similar advantage in mixing experiments, as ligands can rapidly and more uniformly diffuse throughout the crystal, again allowing defined reaction intermediates to be recorded at different time delays30,31,32.
At Diamond's microfocus beamline I24 both conventional rotation and SSX experiments can be performed. Here a comprehensive protocol for SSX sample preparation and data collection using fixed targets at I24 and protocols for data analysis of serial data at Diamond are presented. While the manuscript and accompanying videos should allow users to carry out a successful SSX experiment at I24, it should be noted that this is a rapidly developing field and approaches are continually evolving. It should also be noted that serial methods are available at other synchrotron sources, including but not limited to Petra III (P14-TREXX), MAX IV (BioMAX)33, SLS (PXI and PXII)34, and NSLS (FMX)35. While the the specifics of serial data collection and processing will differ between sources, the core principles will remain the same. The protocols below should be seen to represent a starting point and a pathway to base camp rather than the summit of what might be achieved.
This protocol assumes the users have a protein or small molecule crystal system, from which a microcrystal slurry on the order of 0.5-2.0 mL with a good density of microcrystals per mL has been produced. Protocols for obtaining crystal slurries have been described previously 36. Many different types of fixed target are available, the most commonly used at I24 utilize a precisely defined silicon chip. In order to differentiate from other chip layouts, below and in the beamline interface this is referred to as an 'Oxford chip'. As previously described the Oxford chip layout comprises 8×8 'city blocks', each containing 20×20 apertures for a total of 25,600 apertures20,21.
1. Preparing and loading a chip
NOTE: The process occurs within a humidity-controlled environment (Figure 1), typically between 80% and 90% or higher relative humidity, to prevent protein crystals from drying out. Once loaded and sealed, crystals can survive for upwards of 24 hours. However, this is can vary greatly between crystal systems. Within the chamber a low powered vacuum pump attached to a loading stage to hold a silicon chip (Figure 1), a silicon chip, a chip holder with polyester foil (Figure 2), a p200 pipette, 200 µL pipette tips, tweezers, filter paper and the protein crystal slurry are required.
2. GUI and setup at the beamline
3. Aligning the chip
4. Setting up data collection
NOTE: Data collection setup will depend on the system being studied, and the experiment to be performed. This can range from the simplest SSX experiment, collecting a low dose structure, to a time-resolved experiment using lasers or rapid mixing to initiate a reaction which will require multiple complete datasets at different time delays. To set up a data collection the following parameters need to be defined.
5. Common data collection methods
NOTE: The following are the key parameters that define the type of experiment being carried out. This section assumes that the other settings from protocol 3 "Setting up Data Collection" have been defined.
6. Data processing
NOTE: Broadly speaking data processing can be divided into three groups based on the urgency with which feedback is required. Fast feedback is required to show if crystals are present and diffract, and if so, in what numbers. This should keep up with data collection. Performing data indexing and integration which can be slower but should still be performed on comparable time scales with data collection. Merging and scaling of reflection intensities into an mtz file for structure solution and the generation of electron density maps represents the final step and can be slower still. Here starting pipelines at I24 for the first two stages only will be discussed, as they are required for real-time feedback to guide your experiment, though note that metrics such as hit-rates and scaling statistics are not a substitute for inspecting electron density, which may provide the only confirmation that a ligand has bound, or a reaction occurred, in crystallo.
Low Dose Data Collection and Series
Low dose (Step 5.1: Scenario 1) and dose series (Step 5.2: Scenario 2) data were collected on copper nitrite reductase micro crystals at I24 and have been published previously 42. All samples were prepared as described in step 1, data collected as per steps 3, 4, and 5, and processed using methods in step 6. In this work a rapid dose series was collected with 20 diffraction images taken at each aperture (i.e., n=20 in the data collection GUI shown above) before moving to a fresh sample. From these data a bimodal distribution of unit cells in space group P213 was identified (a = b = c = 97.25 Å, and a = b = c = 96.38 Å). Identifying and separating these unit-cell polymorphs for processing showed a marked improvement in data quality indicators and revealed two different structures in a flexible loop between residues 189-193 instead of the mixed state observed when processing all data together. Identification of such polymorphs could make all the difference in a delicate time-resolved structural study where only small structural changes are expected. Furthermore, the dose series collected revealed a dose dependent unit cell change in the crystal, with increased dose shifting the population in favor of the larger unit-cell.
Similar work was performed by Ebrahim et al (2019)47, where a dose series (Step 5.2: Scenario 2) was collected from a dye-type heme peroxidase from Streptomyces lividans (DtpAa) to compare low dose structures from SSX (Step 5.1: Scenario 1) with those measured in the same fixed target system using SFX. SFX data were collected at SACLA Beamline BL2 EH3 with a pulse length of 10 femtoseconds and a repetition rate of 30 Hz. The 10 femtosecond pulse duration ensures that dose dependent effects are not present in the SFX data. SFX data were compared to SSX data collected on beamline I24, where 10 sequential 10 millisecond exposures were measured at each sample position (i.e., n=10). The dose dependent migration of a heme iron coordinated water molecule away from the iron was observed, as well as a conformational change in one of the heme propionate groups in the SSX dose series. Although not damage-free like the SFX structure, the dose series allowed the Fe-O bond length of a zero-dose dataset (ferric heme) to be extrapolated, with this agreeing within experimental error with the value obtained from SFX.
The serial crystallography data collection methods described here can also be easily adapted to provide new sample environments to, for example, study anaerobic protein structures at room temperature. As outlined in Rabe et al 2020 48, loading a 'sheet-on-sheet' sample, or 'chipless chip', with different sealing films in an anerobic chamber enables room temperature collection of structural data from dioxygen-sensitive samples.
Although the following representative results were not collected at Diamond Beamline I24, these methods have been developed in close collaboration between facilities in the iNEXT program to work towards standard methods in serial crystallography method development. Beamline I24 offers, or will soon offer, equivalent collection methods to those described below to perform such experiments using the methods described in the protocols above.
Pump Probe: Rapid Mixing
Rapid mixing SSX has been performed at beamline T-REXX at PETRA III by Mehrabi et al (2019) 28 using a piezo driven droplet injector to initiate reactions on fixed targets. This work presents a proof of principle on chip mixing experiment binding GlcNac3to lysozyme microcrystals, with binding occurring within 50 ms of a 75 pL drop being applied to the sample. This study was followed up with a 7-structure time-resolved series of xylose isomerase activity, demonstrating glucose binding within 15 ms and the formation of an open ring conformation in the glucose molecule after a 60 second time delay. An equivalent setup for droplet injection is currently under development for use on I24.
Pump-Probe: Light Activation
A light activated pump-probe serial experiment is presented in Schulz et al (2018) 49. Fluoroacetate dehydrogenase was soaked with photocaged fluoroacetate and pumped with 320-360 nm laser light to produce structures at 4 time points (t=0, 30, 752, and 2,052 ms). The resting state structure (0 ms) shows an empty active site, with the exception of a few water molecules, and equivalent density between the cap domains of both protein subunits. 30 ms and 752 ms after light activation a significant reduction in electron density can be observed in the cap domain of subunit B relative to subunit A. The reduction in electron density in the cap domain of subunit B coincides with the appearance of fluoroacetate in the active site of subunit A at 752 ms. The final dataset at 2,052 ms shows further structural rearrangement of the ligand, suspected to facilitate the correct geometry for SN2 attack, and potential formation of an intermediate state in the reaction. On I24, a portable Pharos laser system which is tunable from 210-2500 nm providing femtosecond pulses can be used for light-activation. Initial experiments showed the successful activation of a photocage using 308 nm excitation with binding of the released ligand to the target protein observed. At the time of writing integration into the beamline personnel safety system is ongoing and routine user experiments are anticipated in early 2021. For experiments when less intense pulses of light are required, light-activation with TTL controlled LEDs has been performed successfully.
Figure 1: Sample loading equipment in place at Diamond Light Source. The set-up consists of a vacuum pump (a), glove-box (b), and humidifier (c). Within the glove-box vacuum pressure is used to act on a chip loaded with crystal slurry held in a sample block (d) attached to a Büchner flask (e, green arrow), via a pressure regulator (f, yellow arrow) attached to a stopcock (g, blue arrow). Humid air is pumped into the tent via plastic tubing attached to the humidifier (h), and measured using a hygrometer (i). Components are held in place using clamp stands (j). Please click here to view a larger version of this figure.
Figure 2: Sample holders. They utilize a metal O-ring (a) to clamp polyester film onto a top (b) and bottom (c) half, with the bottom half sporting magnetic mounts (d) that are used to attach the sample holder to the sample stages. The polyester film (6 µm (e) or 3 µm (f)) as well as rubber O-rings (white arrows) prevent a crystal-loaded chip from drying rapidly in a sample holder which is closed tight with hex bolts (g). Chips are cleaned using sequential 15-minute baths in dH2O, 1 M HCl, and dH2O (h). Please click here to view a larger version of this figure.
Figure 3: Data collection GUI for fixed target data collection at I24. (a) Shows the main interface used for aligning chips and defining data collection parameters, (b) is the mapping lite interface used for defining sub-regions of a chip for data collection and (c) is an interface for defining parameters for laser illumination. Please click here to view a larger version of this figure.
Figure 4: The process of mounting a chip holder onto the stages as described in Step 3, point 1. Please click here to view a larger version of this figure.
Figure 5: Chip alignment. A chip is aligned by clicking on three fiducial markers on the chip shown in (a). Views of fiducials 0, 1 and 2 through the beamline on-axis viewing system are shown in (b), (c) and (d). Please click here to view a larger version of this figure.
Figure 6: Auto-processing results displays launched as described in step 6.1. An updating hit-rate plot is displayed (a, inset). If a 'hit' is clicked on the corresponding diffraction image is displayed in dials image viewer. The hit-rate for the current data collection is shown (29.6% in this example). Panel (b) shows an example of a window showing current indexing and integration rates for data collected so far during the visit that updates in real time. Please click here to view a larger version of this figure.
Figure 7: More in-depth data analysis. Visualization of unit cell parameters can reveal polymorphs (a). Average unit cell parameters are calculated; however, this does not yet extend to individual averages for polymorphs. Visualization of a small subset of data (data shown are a subset of 793 copper nitrite reductase crystals from the data described in Ebrahim et al 2019) is often sufficient to reveal trends. 2-D plots of useful parameters can also be produced to reveal variations that arise due to loading or dehydration effects that could be addressed for upcoming data collections (b). Stereographic projections can reveal the presence, or absence, preferred orientations feeding back into the loading protocol (c). Please click here to view a larger version of this figure.
Serial synchrotron data collection is a relatively new technique at MX beamlines, bridging the gap between the ultra-fast data collections currently being performed at XFELs and traditional synchrotron-based MX. This manuscript aims to give an overview of how to successfully collect fixed target serial data at beamline I24, Diamond Light Source for low dose, dose series, and time-resolved experiments. As with standard crystallography, sample preparation is a major bottle neck in structure solution. SSX is no different, and preparation of a homogenous crystal slurry in sufficient quantities has not yet benefited from several decades of study and refinement like the growth of single large protein crystals has. However, preparation of these slurries is outside the scope of this paper and has been summarized elsewhere36. The critical step in the approach described here involves the careful use of the available sample using easy to use GUI interfaces (step 3) and automated data processing pipelines (step 6) to inform the chip loading (step 1) and how an experiment should proceed.
The fast feedback pipeline is a powerful tool that allows users to assess initial hit rates during data collection to inform subsequent chip loading protocols for successful data collection. When faced with a low hit rate (<5%), users risk collecting incomplete data and/or wasting beamtime with additional collections. In this case, sample could be pooled, concentrated by gentle centrifugation, and/or larger volumes could be loaded in step 1.5. A higher hit rate is generally favorable, however, there is a point of diminishing return where overloading leads to multiple crystals in the same well. DIALS is capable of dealing with multi-lattice diffraction data50, but a greater concern than indexing and integration is the detrimental effect crystal grouping can have on the even activation of crystals by laser light or rapid mixing for precise time resolved experiments. Particular care should therefore be taken to avoid overloading fixed targets for time resolved experiments.
The indexing and integration processing step produces a plot with the central cross representing the beam direction, each point representing the direction of the hkl 001 reflection of individual lattices, and the outer ring of the circle representing a rotation of 90° away from the beam axis. This will show if your crystals have a preferred orientation, which may impact data completeness and indicate the need to collect more data or vary the loading protocol. In the left-hand panel of Figure 7c, the effect of overloading a chip with HEWL crystals is shown. As apertures fill with more crystals, they stick to the angled walls of the apertures rather than wedging at the base in a random orientation. The two orthogonal ellipses are a result of crystals lying on the internal walls of the chip which are at ~35° to the beam direction. This reduces the volume of crystals loaded, reduces the hit rate, and dramatically reduces the fraction of crystals lying in these preferred planes.
It should be noted that other serial approaches are available at I24, such as LCP extruders and microfluidic chips. These use similar GUIs and the same processing pipelines so much of the above will remain applicable even if a different technique is used. A number of serial approaches exist for both SSX and SFX beyond the fixed target approach described here, each has certain advantages over the other depending on the experiment to be performed and the beamline used for the experiment. As serial approaches are evolving rapidly it is advisable to check the beamline webpages (https://www.diamond.ac.uk/Instruments/Mx/I24.html) for recent updates and talk to beamline staff at as early a stage as possible when planning beamtime. Access to I24 for standard and serial experiments is free at point of use. For UK and EU users travel and accommodation costs are partly covered through iNEXT Discovery.
This work was supported by the iNEXT-Discovery (Grant 871037) funded by Horizon 2020 program of the European Commission.
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