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10:32 min
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April 23rd, 2021
DOI :
April 23rd, 2021
•0:04
Introduction
1:15
Crystal Harvesting
1:58
Loading of SH from Combipuck to the Transfer Block
3:44
Loading of Transfer Blocks into the Vacuum Vessel
8:08
Results: Diffraction Images and Structure Solution of Thaumatin
9:41
Conclusion
Transcript
Long-wavelength macromolecular crystallography exploits the anomalous signal from light atoms natively present in proteins and nucleic acids. This technique is used to experimentally solve the crystallographic face problem and to determine the identity and location of these elements. Beamline I23 at Diamond Light Source is a unique synchrotron instrument optimized for experiments at long wavelengths up to five angstrom.
It allows access to the absorption edges of elements of high biological relevance, such as calcium, potassium, chlorine, sulfur, and phosphorus. Due to the significant absorption of x-rays by air in the long-wavelength regime, these experiments are performed in a vacuum environment. To keep samples at cryogenic temperatures inside the vacuum environment, the crystals are mounted on dedicated, thermally-conductive sample holders.
The cryogenic sample transfer from liquid nitrogen to the vacuum end station is very similar to the techniques used in cryo-electron microscopy. This protocol presents the procedure for transferring crystals into the vacuum environment using the tools and equipment developed at Diamond Light Source. Begin by separating the lid from the base of the CombiPuck such that the sample holders remain attached to the base and vials are retained in the lid.
Immerse the lid with vials in liquid nitrogen, then attach a sample holder and adapter to a magnetic wand and harvest the crystals. Flush cool each sample directly into the CombiPuck, noting the sample position. To close the puck, use a puck wand to attach the base to the lid.
Transfer the CombiPuck from liquid nitrogen to the dry shipper or storage. Place the base of the block puck already populated with empty transfer blocks onto its support base in a foam container, then fill with liquid nitrogen. Then, place the CombiPuck in the foam container filled with liquid nitrogen, making sure that the base of the puck is secured to the magnetic holder inside the foam container.
Pre-cool all necessary tools in liquid nitrogen. Then, separate the lid from base using the puck separator tool on the high setting, such that the base remains attached to the magnetic holder, exposing the sample holders inside liquid nitrogen. Place the separator wand over the sample holder and adapter as far down as it can go, making sure the wand is vertical.
Move the small lever on the separator wand down with your thumb until it clicks to secure the sample holder inside, and pull the sample holder from the adapter. Lower the separator over the desired transfer block position, making sure one of the three prongs fit inside the central hole of the transfer block. Release the sample holder by moving the lever back up.
To load samples into the next transfer block, use the carousel key tool to rotate an empty transfer block into the horizontal position. Once all sample holders are transferred, close the block puck by placing the lid in liquid nitrogen. Wait for the temperature to equilibrate, and then fit the lid over the base and lift gently to release from the carousel.
Attach the shuttle to the station. Open the nitrogen gas and air valves and ensure that gases are flowing. Then, switch on the CTS.
Cool both the bath and the shuttle with liquid nitrogen. Place the supplied funnel in the filling port on the shuttle and slowly pour liquid nitrogen into the funnel while monitoring the level on the screen. Stop when the indicator turns from red to blue.
Fill the bath with liquid nitrogen using the funnel. Transfer a block puck from liquid nitrogen to the CTS bath using the attached puck separator tool. Remove the lid of the block puck and close the lid of the CTS bath.
To introduce a transfer block into the shuttle, unlock the shuttle handle by rotating 90 degrees clockwise and advance it towards the bath so that the guided track on the handle enforces the correct path of travel. Once the block cover is cooled, advance the handle to introduce the block into the shuttle. To lock the transfer block onto the shuttle, rotate the handle 180 degrees clockwise.
Retract the handle to the original back position, and lock it in place by rotating 90 degrees counterclockwise. Press Close Shuttle Valve Pump on the display screen to start shuttle evacuation. Once the messages Shuttle ready to move and Do not move rod, valve closed are displayed on the touch screen, press the lever underneath the shuttle and carefully lift it using the handle at the top.
Carry the shuttle to the airlock on the vacuum end station in an upright position and attach it. Select an empty block position within the vessel by pressing the corresponding button on the touch screen and moving the sample hotel to the correct loading position. Once a sample hotel is in position, press the Open button to initiate the vacuum interlock sequence.
After the sequence is complete and the status changes to Airlock open, block in shuttle, twist the handle 90 degrees clockwise to unlock the rod. Gently push the rod into the vessel so that the guided track enforces the correct path of travel towards the sample hotel. Slowly insert the transfer block into the hotel using the video feed displayed on the screen for guidance, ensuring that the block position icon on the touch display is activated.
Once activated, rotate the handle 180 degrees counterclockwise to release the transfer block and pull the rod out of the vessel. Once fully retracted, rotate the handle 90 degrees counterclockwise to lock the rod. Press the Close button to close the end station vacuum valve and vent the space between the shuttle and the vessel to atmospheric pressure.
Remove the shuttle when the airlock display shows the status OK to detach. Return the shuttle to the CTS bath and press Open Shuttle Valve to evacuate the shuttle before loading the next sample block. Once the traffic light is green and the message OK to move rod appears, the next transfer block can be introduced to the shuttle.
To prepare the next transfer block, rotate the block puck inside the bath. Push the built-in rotation key on the top of the acrylic lid down into the lock in the center of the block puck. While holding it down, turn the key to position the desired transfer block in the pickup position.
Once all blocks have been transferred, ensure the shuttle valve is open, press the Bake button on the touch screen, select both Bath and Shuttle, then press Start Bake. The long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source can access the absorption edges of elements of high biological importance, such as calcium, potassium, chlorine, sulfur, and phosphorus, giving enhanced anomalous signal which can be used for phasing or locating these elements within macromolecules. Diffraction data were collected from a single thaumatin crystal at wavelength 2.75 angstrom, chosen as a compromise between increased anomalous signal and sample absorption effects at longer wavelengths.
The vacuum setup ensures that only x-rays scattered by the sample reach the detector, providing low background around the Bragg reflections. The dataset yielded very strong anomalous signal, facilitating structure solution by the automatic phasing pipeline CRANK2. The high quality of the resulting electron density map enabled successful automatic model building, with correct placement for 100%of the amino acid sequence of thaumatin.
The 16 cysteine residues within thaumatin form eight disulfide bridges, which are all clearly visible in the electron density map. Since in-vacuum long-wavelength protein crystallography is a new field, we have developed new sample handling tools and equipment. This protocol guides users in safely transferring samples into the vacuum end station on beamline I23 at Diamond Light Source.
The vacuum environment opens unique opportunities to perform diffraction experiments in a wavelength range not accessible at other beamlines. Long-wavelength x-ray crystallography enables experimental phasing of macromolecules directly from native crystals. It also allows unambiguous identification of ions bound to the molecules.
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.