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![[Figure 1]](jm5017fig1mag.jpg)
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Figure 1
Overview of different structure-determination pipelines. Comparison of classical crystallography methods with the in cellulo pipeline. An arbitrary colour scale ranging from green (least demanding) to red (most demanding) indicates the relative level of skills and/or resources required for each step between the three workflows. Steps that are identical in all workflows are shaded in grey. For the in vitro crystallization pipeline (top row), the protein is expressed in large amounts, purified and used to find and optimize crystallization conditions. Crystals are then cryoprotected, harvested with nylon loops and flash-cooled by immersion in liquid nitrogen. In the in vivo crystallization approach (middle row), crystals are grown in the cells that express the protein, bypassing protein purification and the crystallization steps. In the classical microcrystallography approach, cells are lysed and crystals are purified by centrifugation. After mounting on a support, typically a grid mesh, crystals are further incubated with an appropriate cryoprotectant solution, the excess liquid is removed and the support is then flash-cooled. Once the support has been loaded onto a goniometer, each crystal has to be accurately centred in the X-ray beam to ensure that it is properly exposed. In an in cellulo pipeline (bottom row), crystals are produced as in the in vivo approach, but the host cells are not lysed. Instead, crystal-containing cells are sorted by flow cytometry, stained with trypan blue and mounted on a support. No cryoprotectant solution is required. Cell sorting is indicated in orange since it is straightforward and inexpensive if available through a shared instrument/facility, but otherwise requires additional resources and training. Once at the beamline, the stain and the larger size of the cells compared with crystals facilitate the identification of crystal-containing cells and alignment with the beam. The image of the automated liquid-chromatography system is courtesy of GE Healthcare AB, Uppsala, Sweden. |
![[Figure 1]](jm5017fig1.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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