Diffraction data from the 5c8* apo crystals at pH 5.5 were collected at the Berkeley Center for Structural Biology (BCSB) from the Advanced Light Source (beamline 8.2.2) on an ADSC Q315R detector, 5c8*/CD40L complex diffraction data were measured at the Stanford Synchrotron Radiation Lightsource (beamline 9-2) on a Pilatus 6M detector and all other data were obtained at the Argonne National Laboratory Advanced Photon Source (beamline 19-ID) on a Pilatus 6M detector. Crystals were flash frozen in liquid nitrogen prior to data collection at 100 K. Data were indexed, refined, integrated, and scaled using the HKL200017 or MosFLM18 (link) package. The structure of 5c8-WT was solved by molecular replacement using Phaser19 (link). Initial searches using the entire 5c8 Fab coordinates from the complex of 5c8 with CD40L (RCSB Protein Data Bank ID 1i9r) as the input model proved unfruitful. Splitting 5c8 in half and searching sequentially for two ensembles, the first consisting of the heavy and light chain constant region and the second the variable domain, gave a reasonable solution. Structures of the subsequent 5c8* apo variants were solved using the 5c8-WT structure as a search model. The structure of the complex of 5c8* with CD40L was determined by molecular replacement using Phaser with PDB entry 1i9r as the search model. All models were refined using Refmac5,20 (link) and model building was carried out with the program Coot.21 (link) A chemical description for 7-HCAA was created using Sketcher from the CCP4 program suite,22 (link) and the geometrical restraints used in refinement were taken from the bond lengths and angles reported for the crystal structure of umbelliferone.23 All structural figures were made with the PyMOL molecular graphics software.24