Published: April 23rd, 2021
Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.
Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å.
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools.
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.
Long-wavelength X-ray diffraction is used to harness the anomalous scattering properties of specific light atoms natively present in macromolecules. This helps to solve the crystallographic phase problem and to unambiguously confirm the identity and location of such elements within macromolecules. While in the early days of macromolecular crystallography, de novo structures were solved by multiple isomorphous replacement1, with the advent of tunable X-ray beamlines at synchrotrons, experimental phasing based on multi-wavelength and single-wavelength (SAD) anomalous diffraction techniques have become the dominant methods2. Both methods have historically relied on the isomorphous or anomalous signal from heavy metals, which need to be artificially introduced into the crystals by co-crystallization or crystal soaking3. The trial-and-error approach and unpredictable outcome can make these experiments frustratingly time-consuming. The incorporation of seleno-methionine during protein expression4 is a very elegant way to overcome these limitations and exploit anomalous diffraction at short wavelengths, although it can be very challenging in eukaryotic protein expression systems.
Long-wavelength MX is extremely appealing for structure determination by native SAD experiments5,6 due to the convenience of using crystals directly from a successful crystallization trial without further treatment. Additionally, access to the absorption edges of elements of high biological importance, such as calcium, potassium, chlorine, sulfur, and phosphorus, opens the opportunity to directly identify the positions of these elements in macromolecules7,8,9,10. At medium and low resolution, element assignment based on the 2Fo-Fc electron density and chemical environment can be difficult, particularly for elements with similar number of electrons or weakly bound ions with partial occupancies. These ambiguities can be resolved by collecting data below and above the absorption edge of the element of interest and interpretation of the resulting model-phased anomalous difference Fourier maps11,12. Locating sulfur atom positions in these maps can also aid model-building into low-resolution electron density maps13. The absorption edges of these light elements are observed at wavelengths between λ = 3 and 6 Å (see Figure 1, top). This wavelength range has been well beyond the capabilities of any synchrotron MX beamline, and efficient operation in this range requires overcoming several technical challenges, as outlined below.
Beamline I23 at Diamond Light Source, UK, is a unique instrument, specifically designed to facilitate long-wavelength MX experiments, tunable in a wavelength range between λ = 1.13 and 5.9 Å (energy range between E = 2.1 and 11 keV). By operating in a high-vacuum environment14, air absorption and scattering are eliminated, consequently enhancing the efficiency of diffraction experiments and the signal-to-noise ratio. A large vacuum endstation encloses all the components of the sample environment, including the semi-cylindrical Pilatus 12M detector, a multi-axis goniometer, the on-line viewing and collimation systems, as well as the bespoke equipment for sample transfer and storage (Figure 2). Each piece of equipment has been optimized to ensure that the best-quality long-wavelength data can be collected. The curved Pilatus 12M detector can collect to diffraction angles of 2θ = ±100°, resulting in sufficiently high-resolution diffraction data even at longest wavelengths (Figure 1, bottom). The 120 detector modules have been specifically selected for low-energy compatibility and calibrations for an additional ultra-high gain mode have been provided.
The lowest possible detector threshold is 1.8 keV, leading to increased corner and edge effects for energies lower than 3.6 keV and compromised data quality at the longest wavelengths, particularly for low mosaicity crystals, can be observed. This effect in combination with the decrease in the detector quantum efficiency15 needs to be taken into consideration when planning an experiment. The multi-axis goniometer enables reorientation of crystals to allow for data collection strategies that maximize the quality and strength of the anomalous signal, as well as the completeness of the anomalous data collected. Sample absorption is a limiting factor for the experiments, particularly at longest wavelengths. Absorption corrections, as implemented in commonly used MX processing software packages16,17, are working well to wavelengths around 3 Å. Longer wavelengths will require analytical absorption corrections based on tomographic reconstructions18 or laser ablation to remove non-diffracting material and cut the crystals into well-defined shapes19. The latter will also assist in reducing the size of larger crystals as X-ray diffraction experiments at longer wavelengths are more efficient for smaller crystals14. The challenge of keeping samples at cryogenic temperatures during data collection is addressed by conductive cooling, as using open-flow cold gas stream devices is not compatible with a vacuum environment. Hence, thermally conductive materials, such as copper, are needed for connecting the sample to a pulse tube cryocooler. The stainless steel SPINE standard pins used throughout MX, as well as any other commercially available sample mounts, are not suitable for in-vacuum long-wavelength MX because of their poor thermal conductivity.
The sample holders (SHs) for in-vacuum MX must be an essential part of the heat removal thermal pathway (Figure 3A). As such, they consist of a thermally conductive copper body and pin and include two important features: a strong magnet base to ensure an adequate thermal link to the cold goniometer head, and a sample mount, made from polyimide, to minimize X-ray absorption and scattering20. Efforts were made to ensure that the user experience of crystal-harvesting and flash-cooling is almost identical to that associated with standard MX practices. As the dedicated I23 SHs are not directly compatible with other synchrotron beamlines, a stainless steel adaptor is used for compatibility with the crystal-harvesting magnetic wands and existing goniometer interfaces on other MX beamlines (Figure 3B). The adaptor is also important for making use of the automation facilities on other Diamond MX beamlines, which are based on ALS-type robot gripper heads21 and unipuck-style base layouts22, if sample variation requires fast pre-screening for selection of the best diffracting crystals. The sample preparation and loading protocol can be broken down into two stages:
Stage 1: Harvesting crystals and flash-freezing performed by users in their own laboratories
Following assessment of the project suitability for I23 data collection, sample holders with loops matching the crystal sizes (pre-assembled with adaptors) are sent to user labs for crystal harvesting. To prevent any damage, SHs and adaptors should not be separated and are to be used as one unit for the purpose of fishing crystals with appropriately sized loops using standard crystal-harvesting magnetic wands. As is common in MX, this task is performed manually under the microscope, and crystals are immediately flash-cooled in a foam dewar with liquid nitrogen23. Due to a mismatch of magnetic forces, the SHs are currently not compatible with unipucks. Storage and shipping are realized using combipucks (see the Table of Materials), which are available to users upon request, along with the compatible dry shipper inserts (Figure 3C). These pucks share the same base plate with the widely used unipucks and allow fast pre-screening of samples at other Diamond MX beamlines. Loaning this equipment to users is currently the best arrangement, until the bespoke sample holders are commercially available. Transport to the beamline requires the standard dry shippers used in the MX community.
Stage 2: Transfer of cryo-cooled samples into the vacuum endstation
Once the samples arrive on the beamline, they are prepared for transfer into the vacuum endstation. This involves removal of SHs from combipucks and separation from adaptors. Introducing biological samples to vacuum is routinely performed in the field of cryo-electron microscopy. Some of the well-established concepts were adapted for the I23 sample transfer. In short, SHs are transferred under liquid nitrogen onto transfer blocks (Figure 3D). These blocks have excellent thermal conductivity and a significant thermal mass, preventing the crystals from reaching the glass-transition temperature when in vacuum. Up to four blocks, with a capacity of four samples each, are loaded under liquid nitrogen into a block puck (Figure 3H), which is used either for transferring samples to the Cryogenic Transfer System (CTS) or for storage in liquid nitrogen dewars between experiments.
The Cryogenic Transfer System developed at Diamond Light Source comprises of two sub-assemblies, the Sample Station and the Shuttle (Figure 4A). The Sample Station consists of a liquid nitrogen bath for temporary storage of protein crystals and has specific features to ensure safety and allow a user-friendly experience (Figure 5). The CTS is controlled by a programmable logic controller via a user-friendly touchscreen interface. The Sample Station has light-emitting diodes built in for better visualization and a set of heaters controlled in close-loop to automate the drying of the liquid nitrogen bath once the samples have been transferred. It also has a variety of sensors to ensure safety and efficient functioning of the system. The Sample Station has bespoke hardware to provide a reliable electrical interface to interact with the shuttle for operations, such as pumping down to rough vacuum for sample transfer, as well as monitoring of liquid nitrogen levels and the temperature inside the shuttle.
The Shuttle (Figure 6) is a portable device used to pick up a transfer block from the Sample Station liquid nitrogen bath and transfer it inside a cryogenic and vacuum environment to the endstation. It includes a liquid nitrogen dewar to keep the samples cold during transfer, liquid level monitoring in the dewar, and a variety of sensors for operation and user safety. The transfer arm is equipped with a magnetic drive and includes machined grooves to guide users in safely loading and unloading transfer blocks into the endstation. Transfer from the shuttle to the vacuum vessel is conducted via an airlock. The airlock is an interface for the shuttle on the endstation used to evacuate the interspace between the shuttle and the endstation, before opening the shuttle and endstation vacuum valves. The pumping and venting sequences are fully automated and can be operated via a large touchscreen with a user-friendly interface (Figure 4C). The current protocol is used to transfer a thaumatin crystal to the vacuum endstation for data collection.
1. Crystal harvesting
NOTE: Use appropriate personal protective equipment: goggles and gloves, where possible.
2. Sample transfer to vacuum
A thaumatin crystal was introduced to the vacuum endstation using the protocol outlined above. Diffraction data were collected at a wavelength of 2.7552 Å (E = 4500 eV) as 3600 images with a rotation increment of 0.1° and 0.1 s exposure per image. The beam size was adjusted to 150 µm x 150 µm and reduced to 10% transmission, with a corresponding flux measurement of 7.1 x 109 photons/s. The choice of λ = 2.7552 Å is based on a compromise between the increase in anomalous signal and sample absorption effects and the decrease in resolution to longer wavelengths. Although not close to the theoretical absorption edge of sulfur (λ= 5.0095 Å), at this wavelength, the imaginary contribution to the scattering factor of sulfur f" is 1.57 e- , a factor of 1.6-2.1 larger compared to wavelengths between 1.7 and 2 Å. The resulting stronger anomalous signals allow successful S-SAD phasing for more challenging projects.
A variety of difficult phasing experiments have already been carried out on beamline I2324,25,26,27, with data collected at this wavelength. While phasing by S-SAD is possible using much shorter wavelengths, this often requires building up anomalous signal through merging data from many isomorphous crystals to reach multiplicity values over 10028. Due to the enhanced anomalous signal at longer wavelengths, most phasing projects solved on I23 only required data from one crystal. A representative diffraction image is shown in Figure 7, left. Data processing using Xia2-3dii29 produced excellent merging statistics, as outlined in Table 1. Figure 7, right, shows part of a representative diffraction image from the thaumatin data set and illustrates the low background surrounding the Bragg reflections, which contributes to the large I/σ(I) values typically observed in the vacuum setup, ensuring that only X-rays scattered by the sample reach the detector.
The maximum achievable resolution of 1.8 Å is due to the detector geometry and the chosen wavelength of the X-ray radiation. The dataset yielded very strong anomalous signal, reflected in the mid-slope of anomalous normal probability parameter of 2.677, facilitating structure solution by the automatic phasing pipeline CRANK2. The high quality of the resulting electron density map enabled successful automatic model building by the Buccaneer30 module within CRANK231, with correct placement for 100% of the amino acid sequence of thaumatin. The phased anomalous difference Fourier map, calculated with ANODE11, reveals 16 very well-ordered sulfur atoms and one sulfur atom from Cys159 with two alternative conformations, as confirmed by the 18 significant heights of the peaks at the positions of the anomalous scatterers in Table 2. The 16 cysteine residues within thaumatin form 8 disulfide bridges, which are all clearly visible in the 2Fo-Fc map (Figure 8).
Figure 1: High-resolution diffraction data from long-wavelength MX experiments. (A) Plot of f" values against energy, indicating absorption edges of light elements accessible on beamline I23. (B) Maximum resolution achievable at the corners of the P12M detector against energy. Abbreviation: MX = macromolecular crystallography. Please click here to view a larger version of this figure.
Figure 2: Horizontal section through the vacuum vessel with all the components of the endstation. Abbreviation: OAV = on-axis viewing system. Please click here to view a larger version of this figure.
Figure 3: Sample handling tools. (A) I23 Sample holder. (B) MX spine-standard pin (left) next to an I23 sample holder with adaptor (right). (C) Combipuck lid and base with I23 sample holders (blue). Block puck lid and base with two transfer blocks (gold). A dry shipper cane, compatible with both combipucks and block pucks, is visible at the back. (D) Transfer block with four I23 sample holders. (E) Key tool used for rotation of the block puck base. (F) Separator wand. (G) Puck separator tool with two arrows showing the high and low settings. (H) Block puck base with four empty Cu blocks. (I) Lid for the block puck. (J) Foam container with all necessary tools for transferring sample holders from combipuck bases to copper blocks. Please click here to view a larger version of this figure.
Figure 4: Cryogenic Transfer System. (A) CTS Sample station with shuttle attached and the funnels used for filling. (B) A block puck with two transfer blocks positioned inside the CTS. (C) CTS control software touchscreen. Abbreviation: CTS = Cryogenic Transfer System. Please click here to view a larger version of this figure.
Figure 5: Cryogenic Transfer System Sample Station. Abbreviations: LEDs = light-emitting diodes; LN2 = liquid nitrogen. Please click here to view a larger version of this figure.
Figure 6: Cryogenic Transfer System Shuttle. Abbreviations: LEDs = light-emitting diodes; LN2 = liquid nitrogen. Please click here to view a larger version of this figure.
Figure 7: Diffraction images. Left, a diffraction image from the dataset collected on the thaumatin crystal. Right, a diffraction spot surrounded by low-count background pixels. Please click here to view a larger version of this figure.
Figure 8: Structure solution of Thaumatin with automatic pipeline CRANK2 (default settings, no subsequent refinement). (A) Overview of thaumatin with 2Fo-Fc map at 1.6σ (blue) and phased anomalous difference Fourier map at 5σ calculated in ANODE (green). (B) Overview of thaumatin showing only the phased anomalous difference Fourier map at 5σ. (C) Close-up view of a disulfide bridge present in thaumatin with 2Fo-Fc map at 1.6σ (blue) and phased anomalous difference Fourier map at 5σ. Please click here to view a larger version of this figure.
|Data collection wavelength (Å) (energy (eV))
|Number of images x wedge size (°)
|3600 x 0.1
|Unit cell constants
|(a = b, c) (Å)
|(α = β = γ) (°)
Table 1: Data collection and processing statistics for Thaumatin at 2.755 Å wavelength at beamline I23, DLS. For resolution, completeness, Rmerge, Rmeas, Rpim, CC1/2, I/σ(I), and multiplicity, high-resolution shells are shown in parentheses. Abbreviation: DLS = Diamond Light Source.
|Peak height (sigma)
Table 2: Anomalous difference Fourier map peak heights as calculated by ANODE using the phased and automatically built model from CRANK2.
The current protocol has been developed to comply with the sample preparation requirements for in-vacuum long-wavelength MX experiments on beamline I23. It has been in use on the beamline for the past year and has contributed to the successful completion of multiple projects. As indicated by the results presented here, the protocol enables a safe and reliable transfer of samples to the vacuum end-station while preserving their diffraction quality. It is an important aspect for the beamline operation and will be accompanied by in-person user training by beamline staff. Some of the steps are worth being highlighted as critical to the successful and safe completion of the procedure: the transfer of samples from combipuck bases to sample blocks requires accuracy and attention to avoid damaging samples (see step 2.1.4); monitoring of the liquid nitrogen level at all stages is important to prevent samples being exposed to air or being in close contact to parts that are not properly cooled (2.1.3 and 2.2.2); waiting until the Close sequence (2.2.14) is completely finished, before removing the shuttle from the endstation (2.2.15), to avoid degradation of the endstation vacuum.
The conception of the protocol was initiated together with an engineering effort aimed at developing purpose-built equipment for the transfer of protein crystals to the vacuum environment. The final products of this project were the CTS and the associated sample handling tools described above. The CTS is a significant improvement on its predecessor, the Leica EM VCT10014, and removes multiple limitations, such as the lack of sample shielding and vacuum environment during transfer, ice build-up inside the liquid nitrogen bath, and the absence of an intuitive user interface and safety features. Additional features of the CTS that improve the user experience are temperature and liquid nitrogen level monitoring inside the shuttle and sample station, a larger capacity bath accommodating four blocks simultaneously, rather than one, and a self-guided mechanism for the shuttle operation. The CTS is fully integrated into the beamline control system with a user-friendly touchscreen interface and enhanced vacuum and mechanical safety when interfacing with the endstation.
Beamline I23 is the first long-wavelength MX synchrotron instrument of its kind and, as such, introducing protein crystals to a high-vacuum environment and storing them at cryogenic temperatures, has required considerable efforts. Improvements to the sample preparation tools and protocol, as well as efforts to streamline processes, are on-going. As part of user support, beamline staff are always available to assist with troubleshooting. An example of one such scenario would be issues that compromise the integrity of the vacuum system, leading to difficulties in attaching or removing the shuttle to/from either the CTS or endstation airlock. Different levels of tests are performed on a weekly and daily basis, and user training will cover additional checks to avoid potential failures, like visual inspection of the O rings on the interfaces the shuttle attaches to. While the vacuum environment opens the opportunity to perform diffraction experiments in a wavelength range not accessible at other beamlines, the additional transfer step reduces the overall sample throughput.
The manual transfer with only four samples per transfer block and up to five blocks inside the vacuum vessel limits the total capacity to 20 samples. Hence, for projects with a large sample to sample variability, samples should be pre-screened at the Diamond high-throughput beamlines, and then only the most promising samples should be transferred for the subsequent optimized long-wavelength experiment. While the sample holders and the transfer blocks are unchanged from their initial introduction a few years ago, the handling tools presented here are all new developments. The I23 dedicated sample holders are immutable due to their role in the cooling concept for the beamline. As such, the design of the sample handling tools aimed to create a link between this new type of holder and standard commercially available tools that the MX user community had adopted for a long time, such as combipucks, crystal harvesting wands, and the dry shipper transport system. Their design involved significant consultation with the user community and required several iterations to complete. The equipment, tools, and protocol presented here represent a simple and robust system for the transfer of user samples for experiments at beamline I23 at Diamond Light Source. This instrument for in-vacuum long-wavelength macromolecular crystallography opens new opportunities for structural biology.
We would like to thank Adam Taylor, Adam Prescott, Ken Jones, Arvinder Palaha, and Kevin Wilkinson for their support in the development of the Cryogenic Sample Transfer System (CTS). This work was funded by iNEXT-Discovery (Grant 871037) funded by Horizon 2020 program of the European Commission.
|single-photon-counting X-ray detector
|Cryopucks used for cryogenic storage and transport of I23 samples holders and samples
|Crystal-harvesting magnetic wand
|Used for harvesting crystal
|Dry Shipper (CX100)
|Used for cryogenic storage and transport of I23 samples holders and samples
|Dry shipper insert (CombiPuck Transport Cane)
|Used for cryogenic storage and transport of I23 samples holders and samples
|sample mount made of Kapton polyimide
|acrylic lid with built-in rotation key
|Used for production of thaumatin crystals by vapour diffusion
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