Understanding the three-dimensional structures of proteins underpins drug design biotechnology and our understanding of many different diseases. VMXi determines protein structures without cryocooling, so much closer to their natural temperatures. VMXi also measures diffraction data from protein crystals while still in their crystallization plates, avoiding any damage from mechanical handling.
In the future, we really want to exploit the incredible throughput of VMXi to explore large populations of crystals to identify lots of different structural states rather than the single structure people often obtain now. We also want to push towards drug development and carrying out fragment screening and optimization within crystallization plates, a new approach that shows enormous promise. To begin, produce crystals within NC2 plates.
Then log into the ISPyB system using the provided Diamond Fed ID and select Proposals. Scroll the list or type the proposal number into the search bar to locate the desired proposal. Once the proposal is found, choose Shipment from the dropdown menu under the proposal number.
The shipment's window will open, displaying all shipments related to the proposal. To create a new shipment, click Add Shipment located on the top right corner of the screen and provide a name for the shipment. Then select Automated/Imager and click the Add Shipment button at the bottom left of the screen to confirm the shipment.
Navigate to the shipment window and click on Add Container, which will prompt the add container page to appear. Now, choose an appropriate plate type from the container type dropdown menu. Input a barcode and container name as per instructions from the Beamline staff for the experimental plates.
Select the VMXi 20 degree Celsius imager from the Requested Imager dropdown menu, and then select the Fibonacci imaging schedule from the Imaging Schedule dropdown menu. From the Crystallization Screen dropdown menu, select the crystallization screen, then choose the correct username from the Owner dropdown menu. Click View and enter the accurate contact address in the email box.
Enter the details about the plate in the comments box. Select the relevant sample from the Protein dropdown menu and use the acronym registered in UAS and approved by Diamond within the experimental proposal. Enter the same name in the sample name box, and leave the remaining boxes empty.
Click the Plate icon to replicate the sample over the whole plate and populate the whole container with green squares. Click Add Container at the bottom of the page to register the plate. Transfer the plate to the appropriate imager where it'll be stored and imaged.
A visit will be generated when the container is stored in the imager, and the user will receive an email with a link to the plate and its images. To view the imaging results, navigate to the proposal of interest, select Containers from the dropdown menu under the proposal number and view the list of available containers. Select the filter Plates if other sample holder types are present.
To narrow the search, use the My Containers option to display only relevant containers associated with the current user ID.Place the cursor over the line and left click to select the desired container. Then a new window showing an overview of the plate will appear. Select a drop on the left-hand side of the plate representation to view its recent image.
Navigate between drops using arrow keys or select them individually using a mouse or cursor. To manually add points for data collection, press the Mark Point button. Position the cursor over the desired point of interest.
Make the selection and wait for a red cross to appear. Once all the points are marked, click the Finish button. Click the Mark Region button to add regions for data collection with grid scans.
Click the top left hand point, drag it down and to the right to create a region that will be scanned on the Beamline. Click the Finish button once all desired regions are established. Look for the blue crosses on the drop images, which represent crystalline objects located automatically by CHiMP.
To view the CHiMP assessment for crystallization drops, check Show Auto Scores and choose crystal in the class dropdown. When all points and regions have been marked in the respective drops, click the Prepare for Data Collection button at the bottom of the page. Review the list of preselected or automatically identified samples.
To add individual points or regions, press the button or click the Add Current Page to Queue button to add all displayed samples. Then click the point filter and tick the Select All checkbox to apply the same parameters to all samples in the current queued samples list. Use filters for point, region, manual, and auto.
For regions, choose Grid Scan DMM 10 micron steps 100%transmission. For other point experiments, select the appropriate options from the dropdown menu. For oscillation data collection, select Omega Scan DMM 10 degrees 10%transmission to maximize data collection.
After selecting the correct experimental parameters for all samples, click the Queue Container button. Once the plate reaches the top of the queue, it will be presented to the Beamline for data collection and then returned to sample storage. Then look for an email with a link to access the relevant data.
Select Sample Group Management from the dropdown menu under the proposal number and look for a list of groups if other users have already created them. To generate a new group, click the Create Sample Group button. Click a shipment from the dropdown menu on the Create Sample Group page to see the sample viewer.
Once a container has been clicked, look for a graphic showing the plate overview. Click on the individual drop or the relevant row letter or column number. After selecting all wells associated with an individual group, enter a name for the group in the Sample Group Name box, and click the Save Sample Group button.
Click the View Sample Group button on this page to return to the list of already generated sample groups associated with the proposal and proceed with data processing.
