1.Soaking crystals

1. Take the screening plate (here, an F2X-Entry Screen plate, Figure 2) from the -20 °C freezer and place it onto the bench/table for about 30 min to pre-warm it to room temperature, thus avoiding condensation moisture.
2. Arrange the working place with two closely arranged microscopes and all tools needed (Figure 3A). The materials are listed in the Table of Materials.
3. Choose 3-4 loops of the appropriate size for transfer of the crystals to be soaked and place them close to the microscopes.
4. Fill the glass spot plate cavities with de-ionized or distilled water.
5. Prepare 5 mL of soaking solution.
6. Cut open the bag of the screening plate pre-warmed to room temperature.
7. Remove the lid and the foil from the screening plate, while keeping the plate placed on the bench/table. 
8. Decant the 5 mL of soaking solution in the reagent reservoir.
9. Fill each of the 96 reservoirs with 40 µL of soaking solution using the 12-channel pipette.
10. Place the EasyAccess Frame on top of the screening plate and secure it with the included clamps by sliding them onto the left and right side of the device.
    NOTE: The EasyAccess Frame is a special device for handling multiple crystals, which was developed at the HZB¹⁴. It enables easy access to each well by shifting the movable tiles while protecting the other wells from evaporation.
11. Place the screening plate (incl. the EasyAccess Frame) under the first microscope and the crystallization plate including the crystals to be soaked under the second microscope.
12. Slide open well A1 of the screening plate by moving the respective acrylic glass tile of the EasyAccess Frame either with a finger or the supplied pen tool. 
13. Add 0.4 µL of soaking solution from the reservoir to the fragment containing well (upper left lens) using a fresh pipette tip. Check through the microscope that the drop covers the dried-on fragment, so it can dissolve.
    NOTE: Alternatively, this step can be carried out using a pipetting robot before the assembly of the EasyAccess Frame. This way the soaking drops of all wells could be placed in one automatic procedure. However, the authors recommend adding the soaking solution directly before the soaking step as described to ensure that the fragment solubilizes slowly and in the presence of the crystal. This avoids that the crystal experiences a sudden shock upon transfer to a drop with a high fragment concentration.
14. Under the second microscope, cut open the sealing foil of the crystallization plate at one of the wells that contains the target crystals.
15. Transfer two crystals using an appropriately sized loop mounted on the crystal wand to the well A1 of the screening plate under the first microscope.
16. Wash the loop in the prepared glass spot plate and dry it by gently touching the tissue. Do this after every transfer to avoid cross contamination with fragment containing soaking solutions.
17. Use the microscope to check that the crystals have been properly placed.
18. Move on to the next well (e.g., B1).
19. Repeat steps 1.13-1.18 with all 96 wells of the screening plate until each soaking drop contains two crystals.
20. Remove the screening plate (incl. the EasyAccess Frame) from under the microscope and place it onto the bench/table.
21. Remove the EasyAccess Frame from the screening plate.
22. Seal the screening plate with sealing foil and place it in the crystallization incubator or cupboard, respectively, where the crystals were grown.
23. Incubate for the optimized soaking time. Overnight is usually convenient.
24. (optional) Preparation of approximately 40 apo crystals (i.e., mock soaking)
    1. Take an MRC 3-lens 96-well low-profile crystallization plate and fill two columns with 40 µL of soaking solution per well using the 12-channel pipette.
    2. Place the EasyAccess Frame on top of the crystallization plate and secure it with the included clamps by sliding them onto the left and right side of the device.
    3. Slide open the acrylic glass tile of well A1.
    4. Place 0.4 µL of soaking solution in each of the two left lenses of the well.
    5. Transfer 2-3 crystals to each drop. After each transfer, wash the loop in the prepared glass spot plate and dry by gently touching the tissue.
    6. Move to the next well (e.g., B1).
    7. Repeat steps 1.24.4-1.24.6 until about 40 crystals are ready for incubation.
    8. Remove the crystallization plate (incl. EasyAccess Frame) from under the microscope onto the bench/table and remove the EasyAccess Frame.
    9. Seal the crystallization plate with sealing foil and place it in the aforementioned crystallization incubator or cupboard.
    10. Incubate for the same time as the screening plate.

2.Harvesting crystals

1. Take out the incubating plate(s) from the incubator or cupboard, respectively.
2. Arrange the working place with one microscope and all tools needed (Figure 3B). The materials are listed in the Table of Materials.
3. Prepare a Unipuck foam dewar with 3 Unipuck lids (i.e., sample enclosures) and fill it with liquid nitrogen (LN₂).
   NOTE: Observe the appropriate safety precautions for working with LN₂ (i.e., wear safety goggles and use suitable protective equipment). It is best to get fresh LN₂ several times during the session in order to avoid water condensation in the LN₂ storage can. Through the entire following procedure, make sure the LN₂ level in the foam dewar is always reaching the upper edge of the dewar. Also ensure that the LN₂ is ice-free; frequently replace the LN₂ (e.g., once every 45 min), or latest if ice starts to accumulate. Then, fill the second foam dewar and transfer Unipucks to it. Empty the icy foam dewar and remove residual ice and moisture with the blow dryer.
4. Remove the foil from the screening plate and place the EasyAccess Frame on top.
5. Slide open well A1.
6. Harvest two crystals from the drop and flash-cool them in LN₂ (one by one) by plunging with a fast vertical movement into the LN₂ and then inserting the sample in the proper puck position. Take relevant notes on the sample tracking sheet.
7. Cryoprotection step (if necessary for the target crystals). In such case, perform this step instead of 2.6.
   1. Place 0.4 µL of soaking solution including cryo-protectant on the lower left lens of the well.
   2. Pull the loop with a crystal mounted from the drop in the upper left lens slowly through the solution in the lower left lens while keeping the crystal in the loop, and then flash-cool in LN₂. Harvest two crystals in this way.
      NOTE: In steps 2.6 and 2.7, make sure that the time the crystal is in the loop and exposed to air is kept very short. The plunging (i.e., the vertical drop of the sample in the LN₂-filled dewar) should be performed as fast as possible. This ensures high sample quality and prevention of ice rings in the data. Track the samples (i.e., note if crystals have damages, etc.) to prioritize either duplicate for the following X-ray measurements, use the template for that. Even if crystals have cracks, “hairs” or other defects due to the soaking, they can still be used and should always be harvested. In case crystals broke into several pieces, two of the biggest/best looking pieces should be harvested. Figure 5 shows some examples of how such crystals can look like. All the shown crystals gave still useful datasets in the respective campaign¹¹, underlining that it is worth to harvest crystals after soaking treatment, even if substantial morphologic changes occurred.
8. Go to the next well and repeat steps 2.5 – 2.6./2.7 until all three pucks are filled.
9. Add the Unipuck bases on top of the lids after pre-cooling them in LN₂.
10. Store the Unipucks in storage racks in a transport dewar or storage dewar.
11. Repeat the preceding steps until all the wells of the screening plate have been processed.
12. (optional) If mock-soaked crystals were prepared, harvest them in a similar fashion as described beforehand.
    NOTE: If two crystals for each of the 96 conditions of the screening plate could be flash-cooled, there will be space for 32 mock-soaked apo crystals, to fill up the 14 Unipucks.
13. Store the Unipucks in LN₂ until the measurement.

3.Data collection 

1. Transfer the Unipucks to beamline BL14.1. If SPINE pucks have been used in step 2, transfer them to beamline BL14.2.
2. Carry out standard measurements on the beamline using the specific recommendations given below. Details about the facility and the experiment control program MXCuBE2 have been presented previously^15,16. Figure 4 shows the interior of the experimental hutches of beamlines BL14.1 and BL14.2 as well as an example screenshot of the MXCuBE2 control software at beamline BL14.1.
   1. To maximize time efficiency and throughput, skip the collection of test images. The sample-to-detector distance will be fixed to a value that is suitable for the upper resolution limit of the crystal system determined in earlier experiments. If the data collection strategy was not optimized beforehand, collect 1800 images of 0.2 degrees each with an exposure time of 0.1 s per image. 
   2. Ideally, test the data collection strategy in prior experiments using mock-soaked apo crystals. For higher symmetry space groups, 1200 images or even 900 images (i.e., 240° or 180°, respectively) will already give complete datasets with good statistics, independent of the starting angle of data collection. 
      NOTE: Higher redundancy and finer slicing can yield superior data quality¹⁷. However, using this “enough but not more” strategy proposed here is an excellent trade-off between quality, data collection time, as well as computational requirements for analysis later on. In the described way, 200 data collections in 24 hours are well possible at beamlines BL14.1 and BL14.2. Nevertheless, samples should be prioritized.
   3. First collect diffraction datasets for one sample per fragment condition, based on the prioritization in step 2.6./2.7 (i.e., collect the data for the higher prioritized duplicate).
   4. For those experiments in 3.2.3 where data collection failed, diffraction was lost or severe ice rings occurred, collect data for the second duplicate sample of the respective fragment condition.
   5. Collect diffraction datasets of apo crystals (if prepared according to steps 1.24 and 2.12).
   6. Collect diffraction datasets of the remaining duplicates of each fragment condition. 
   7. In the MXCuBE2 program, match the dataset identifiers of a CFS campaign to the following pattern: <protein>-<library>-[ABCDEFGH][01][0123456789][ab] (e.g., MyProtein-F2XEntry-B05a, where “B05” stands for the well (i.e., the fragment condition in the screen) and the following “a” for the first duplicate.)

4.Data treatment

1. For data analysis of the CFS campaign, use FragMAXapp, a web-based solution to control a multiplex analysis for processing auto-refinement and PanDDA hit evaluation of CFS data¹⁸ (Lima et al. FragMAXapp, unpublished data). In the FragMAXapp version deployed at HZB the following programs/pipelines are available: XDSAPP¹⁹, Xia2-DIALS and Xia2-XDS²⁰, fspipeline⁷, DIMPLE²¹, Phenix LigFit²², PanDDA^13,23. Use a well refined input model of the target protein as input for automatic refinement; otherwise perform meticulous refinement of one high resolution mock-soaked crystal that was collected during the campaign. 
   NOTE: A key element for hit identification is PanDDA. Details are explained in the respective publications^13,23. In brief, PanDDA automatically calculates electron density maps of a set of data sets in a CFS campaign. These are then assumed as non-binding fragment conditions and averaged to generate the so-called ground state model. The ground state model is then used to derive local discrepancies between each electron density map and the ground state map, using voxel-associated Z-scores. Then, for areas of high Z-scores a so called PanDDA-map is created by fine-tuned subtraction of ground state density from the respective map. This largely enhances the visibility of fragment binding events.
2. To maximize the outcome of PanDDA, use a two-step approach. Firstly, performing a PanDDA run (pandda.analyse) with standard settings. Even if mock-soaked crystals have been collected, their identity will not be included as a parameter (which is possible nonetheless) in order to enable an unbiased generation of the ground state model by PanDDA from all available data. Afterwards, the output data is evaluated by the user via a so-called PanDDA inspection in Coot²⁴. Here, hits with relatively high confidence should be noted, concluding the first step.
3. Secondly, re-run the pandda.analyse step excluding the preliminary hits (determined in the first step) from the ground state model via the –exclude_from_characterisation="<list-of-bound-dataset-ids>" command line option. Further details are described on the PanDDA help pages (https://pandda.bitbucket.io/). This way, datasets that are clear hits and thus would obscure the ground state model if included are disregarded. This leads to an improved ground state model and thus to improved results overall. Finally, a thorough PanDDA inspection is performed to complete the hit identification.
   NOTE: FragMAXapp includes also an output option to save the modeled bound states or prepare data for PDB submission, for further detail see FragMAX webpages (https://fragmax.github.io/).