Computational protocol: Crystal Structure of a Human Single Domain Antibody Dimer Formed through VH-VH Non-Covalent Interactions

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Protocol publication

[…] To determine the circular dichroism (CD) profile and thermostability of the sdAbs, proteins were first buffer-exchanged in 10 mM phosphate buffer, pH 7.0, by a SuperdexTM 75 SEC. The peaks for the sdAbs were collected, and the proteins were used for CD analysis. CD spectra were collected from 250 to 200 nm at protein concentrations of 2.5 µM in a 10 mm quartz cuvette with a J-850 CD spectropolarimeter (JASCO, Easton, MD). Data were collected with a band width of 1.0 nm and a scanning speed of 50 nm min−1 with two accumulations of scans to determine the CD profile of the proteins. Under the same conditions but with a single data accumulation, CD spectra were automatically measured at 2°C intervals from 25–91°C to determine thermal denaturation of the protein at a temperature shift speed of 1°C/min. Ellipicity at 217 nm were plotted against temperature and melting temperatures (Tms) were calculated from Boltzmann Sigmoidal equation using GraphPadPrism software. [...] The Gr6 structure was solved by molecular replacement with the program Phaser , using data between 20 and 2.5 Å. The monomer structure of the camelized human antibody fragment cVH-E2 (PDB code 1OL0) was used as the search model and two copies of the molecule were applied in the search. The best solution from Phaser had a log-likelihood gain of 805.3. The model was rebuilt with the program Coot with the guidance of the 2Fo-Fc and Fo-Fc electron density maps. Refinement of the model was carried out with the program CNS without any non-crystallographic symmetry restraints. Multiple cycles of simulated annealing, positional, and individual isotropic B factor refinements against data to 1.6 Å resolution were performed with CNS, alternating with manual model rebuilding in Coot. 6.7% of the data were flagged for cross-validation during refinement. Total of 266 water molecules were included in the final model and final stage of the refinement. The refinement statistics are summarized in . […]

Pipeline specifications

Software tools GraphPad Prism, Coot, CNS
Applications Miscellaneous, Protein structure analysis
Organisms Homo sapiens