Computational protocol: Room temperature structure of human IgG4-Fc from crystals analysed in situ

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Protocol publication

[…] Data were collected at room temperature (293 K) at beamline I03 at the Diamond Light Source (Harwell, UK) from crystals in situ. Small wedges (typically 3°−6°) of data were collected from different crystals, or spatially distinct regions from a single crystal, using an oscillation per image of 0.2°. For multiple datasets collected from a single crystal, the oscillation start angle for each dataset was incremented by 2°. Over 200 partial datasets from 48 isomorphous crystals were collected in this manner. Integration was performed with XDS () within the xia2 package () and further processing was carried out using POINTLESS (), SORTMTZ, AIMLESS () and TRUNCATE () from the CCP4 suite (). Only the first 10 images (2° of data) from each partial dataset that had been successfully integrated with XDS, with Batch Rmerge values of 40% or less, were typically used for scaling, with 129 runs of data finally included. The structure was solved by molecular replacement with PHASER () using protein atoms from PDB: 4C54 as a search model, with residues 325–331 omitted from the model. Refinement was performed with PHENIX (), using the “Optimize X-ray/stereochemistry weight” and “Optimize X-ray/ADP weight” options, and manual model building was performed with Coot (). For both chains of the asymmetric unit, the Cγ2 domain FG loop conformation was validated by inspection of 2Fo-Fc and Fo-Fc electron density maps following refinement with residues 325–331 omitted from the model (). Structure quality was assessed with MolProbity () within PHENIX. Data processing and refinement statistics are presented in . Interfaces were analysed with PISA () and figures were produced with PyMOL (The PyMOL Molecular Graphics System, Version 1.1r1, Schrödinger, LLC). […]

Pipeline specifications

Software tools XDS, xia2, CCP4, PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Diseases Drug-Related Side Effects and Adverse Reactions