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Joint Research Group Macromolecular Crystallography

Guidelines

The objective of a single fragment screening (FS) experiment is to individually soak 96 different chemical fragments into crystals of the desired protein target and to analyze the respective complex structures by X-ray crystallography. In order to maximize the chances for a successful FS experiment, the following requirements should be met at the outset.

To conduct a FS experiment with us please read the guidelines and contact Manfred Weiss or Jan Wollenhaupt. 

Prerequisites:

  1. Reliable and reproducible crystallization conditions should already be established for the desired target. At least 200 crystals are needed for a single FS campaign. Ideally, the crystals should be at least 50 μm in size in each direction.
  2. Soaking and cryo-conditions that yield diffracting crystals should be established. Ideally, the crystals should tolerate DMSO to a concentration of 5 or 10% (v/v). The diffraction properties after the desired soaking time (ideally o/n, but 1h - 48h possible) should be better than 2.0 Å, but certainly not worse than 2.3 Å. If a known substrate or inhibitor is availabe, a positive control binding experiment should be performed prior to attempting a FS experiment. The experiment becomes less cumbersome, if soaking and cryo-protection can be combined into one step.
  3. A proposal for beam time for the FS experiment should be submitted in advance through HZB's electronic user office GATE in order to be able to book beam time for the experiment. Typically, six shifts should suffice. GATE accounts and valid online-safety training is also necessary to access the ring and thus the prep lab, in case soaking experiments are carried out on site. If you need access to our BioLab as well, registration is  necessary  at  least  one  week  prior  to  your  visit. International user experiments have the possibility to apply for funding through the EU Horizon 2020 Project iNEXT-Discovery. For more information, please get in touch.

Checklist:

Please go through the following list of questions carefully.

  • Can >200 protein crystals be produced in one crystallization condition?
  • Are your crystals larger than 50 μm in each direction?
  • Do the crystals tolerate 5-10% (v/v) DMSO in the soaking/cryo-condition?
  • Is the resolution of the crystals after soaking and cryo-cooling typically better than 2.0 Å?
  • Is your cryo-protectant included in the soaking condition?
  • Was the positive control bound to the protein after soaking and cryo-cooling?
  • In case you want to bring crystals, do they still diffract after e.g. several weeks?

If you can answer all questions with YES you are perfectly prepared and ready to start your FS campaign. If you can answer most questions with YES you are still well prepared and almost ready. Also, not all campaigns necessarily need to fulfill all criteria. Please do not hesitate to contact us if you have any questions.

Preparation for the visit:

  1. Loops, vials, bases and pucks for sample mounting and storage need to be provided by you. Please bring a sufficient number of vials and bases as well as sufficient number of loops of appropriate size for your crystals (we recommend the MiTeGen MicroLoops). If you would like to store your samples for data collection after your initial visit, please bring an appropriate number of SPINE/unipucks pucks.
  2. Minimize storage time of the frozen crystals. This is recommended in general but especially if you perform the crystal treatment on site, let the beam time then follow as soon as possible.

During the visit:

  1. F2X-Entry screen or HZB library will be provided by us. However, make sure to use of it in the most economical way. Ideally, plates should be unsealed and used for soaking experiments in one day.
  2. Accommodation is available for BESSY II users at the guesthouse.
  3. Naming of Datasets. To facilitate data processing, refinement and analysis via XDSAPP, fspipeline and PanDDA, please stick to the following naming conventions:

    F2X-Entry Screen and FragXtal screen according to the well identifyers:

        <protein>-<library>-[ABCDEFGH][012][0123456789][abcdef]

        e.g. MyProtein-F2XEntry-B05a

    HZB-library and other libraries according to 3-digit numbering:

        <protein>-<library>-F[0123456789][0123456789][0123456789][abcdef]

        e.g. MyOtherProtein-HZBlib-F085a

    (the letter in the end of the name identifies measurements of the same Fragment, e.g. duplicates)

        For apo datasets please use:

        <protein>-<library>-Apo[0123456789][0123456789]

        (i.e. counting numbers, always two digits)

        e.g. MyProtein-F2XEntry-Apo04 or MyProtein-F2XEntry-Apo13

 

After the visit:

Data treatment and hit finding

To facilitate the analysis of the hundreds of data sets measured in a FS campaign, we provide  automated solutions for data processing, refinement and hit identification. They are all combinded in the FragMAXapp (Lima et al., 2021) interface running on a dedicated server at HZB that will be available to you.

Included in the processing pipelines are several auto-processing pipelines and auto-refinement piplines, among those XDSAPP (Sparta et al., 2016), for data processing. And refinement and hit identification pipeline based on PHENIX (Adams et al., 2010) and COOT (Emsley et al., 2010) that was developed together with the Drug Design Group in Marburg (Schiebel et al., 2016), called fspipeline. Hit identification can be done by inspecting difference density peaks in the browser-based interface of FragMAXapp or  automatically by PHENIX LigandFit. However, the prefered hit identification is done using  Pan-Dataset-Density-Analysis (PanDDA) (Pearce et al., 2017). For that, results of the refinement pipeline(s) are automatically arranged and submitted for PanDDA, in order to efficiently visualize low-occupancy binders (Pearce et al., 2017).