Feb 18, 2025
XChem crystallographic fragment screening
Forked from XChem solvent test
- Peter Marples1,2,3,
- Daren Fearon1,2,3,
- Blake Balcomb1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3ASAP Discovery Consortium
- ASAP Discovery
External link: https://www.jove.com/v/62414/achieving-efficient-fragment-screening-at-xchem-facility-at-diamond
Protocol Citation: Peter Marples, Daren Fearon, Blake Balcomb 2025. XChem crystallographic fragment screening. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnzwxgk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 26, 2024
Last Modified: February 18, 2025
Protocol Integer ID: 104139
Keywords: XChem, FBDD, fragment, drug discovery, fragment screening
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Acknowledgements:
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK
Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot OX11 0FA, UK
Abstract
The XChem Facility at Diamond Light Source provides routine, large scale crystal graphic fragment screening, supporting the entire crystal to deposition process. X-ray crystallography is an essential part to the FBDD toolkit. It is sensitive enough to identify weak binders and directly yields structural information about the interactions on a molecular level. Here we summarise a generic layout of a crystallographic fragment screening protocol.
Guidelines
N/A
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
Loops https://moleculardimensions.com/en/category/mounted-litholoops Codes: Catalog # MD7-131, Catalog # MD7-133, Catalog # MD7-135, Catalog # MD7-138
Safety warnings
Follow all handling warning for the chemicals use.
Use of cryogen liquid nitrogen; cold burns can occur with exposure of skin to very low temperatures, contact with eyes can cause permenant damage, evaporatin liquid nitrogen can cause oxygen depletion, asphixia and death. Wear appropriate footware and PPE when handling including cryo gloves and safety glasses. Use apportiate volumes of liquid nitrogen for the ventialtion of the laboratory while in use.
Ensure centifuge is properly balanced when used.
Before start
Grow any necassary crystal plates in sitting drop plates.
Imaging
Imaging
Image all sitting drop crystal plates required for the experiment in a crystal plate imagers.
Selecting crystals
Selecting crystals
Based on the images, select drops with suitable crystals for the experiment. Fragment dispensing is done in singletons. i.e. one compound per drop of crystals. Ensure enough drops for the total number of compounds required for the experiment.
If you have a targeting system, target the drops for dispensing compound, not directly targeting the crystals directly to avoid damaging crystals.
Fragment dispensing
Fragment dispensing
3h 2m
3h 2m
Based on the results of your solvent test, calculate the % v/v required of compound to dispense into crystal drops. This percentage chosen will now include compound to dispense into crystal drops.
Note
When changing from solvent to compound, the tolerance of crystals often is reduced, so the optimal soaking time may need to be lowered by 5% v/v compared to the solvent test results.
Protocol
NAME
XChem solvent test
CREATED BY
Peter Marples
Take the compound plate containing the fragments and spin the plate in a benchtop centrifuge for 00:02:00 at 200 x g .
2m
Dispense the required volume of compound into the corresponding crystal drop.
Store the plate in the incubator for 01:00:00 to 03:00:00 depending on the results of your solvent test at either 4 °C or 20 °C depending on the crystal growth temperature.
3h
Harvesting crystals
Harvesting crystals
In preparation for crystal harvesting, if cryoprotectant is required add the necessary volume of cryo onto crystal drops just before full soaking time has ended.
Select a tray of the appropriately sized loops for the crystals (35 µm , 75 µm , 150 µm or 300 µm ). Importantly, choose a loop size that matches the size of the crystal as closely as possible to improve the data quality.
Place the pucks in a foam dewar and cool them down with liquid nitrogen.
Safety information
Wear appropriate PPE when handling liquid nitrogen.
If the crystal has survived, mount the crystal in the loop and plunge into the liquid nitrogen, placing the sample into a puck.
If the crystal has been successfully mounted, record the success and continue to the next sample. Otherwise record the failure and continue to the next sample until all samples that can be harvested have been mounted.
Place lids on the pucks and store.
Send the pucks to be collected.
Protocol references
Douangamath, A., Powell, A., Fearon, D., Collins, P. M., Talon, R., Krojer, T., Skyner, R., Brandao-Neto, J., Dunnett, L., Dias, A., Aimon, A., Pearce, N. M., Wild, C., Gorrie-Stone, T., von Delft, F. Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source. J. Vis. Exp. (171), e62414, doi:10.3791/62414 (2021)