Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

Phage display library construction Phage display libraries of the switch region of the heavy chain of anti-Asf1 Fab12E were created through Kunkel mutagenesis. A series of libraries to the switch residue sequence (S 112 SASTKG 118 ) were created. Libraries included 1, 2 or 3 residue deletions in the switch region (residues 112–117). Additionally, a library with a single switch residue deletion coupled with Gly/Pro diversity at position 118 were created. In total, four libraries were generated, where the largest sub-library (V 111 X 5 (G/P) 118 P 119 ) had a theoretical size of ~1×10 7 .

Fab Expression and Purification

Fabs were expressed as previously described ( 34 ). Briefly Fabs variants were sub-cloned into the pSFV4 vector. Protein was expressed using E. coli BL21 cell line where cells were grown in 2xYT and expression was induced at OD600 ~0.6. Induction proceeded for 5 hours at which point cell pellets were harvested. Fabs were purified as previously described using ProteinG-A1 resin for single-step purification [ 35 ]. Surface plasmon resonance Surface plasmon resonance affinity determination was performed by a BIAcore-3000 instrument. Antigen was immobilized via a C-terminal 6× His tag to a Ni-NTA chip (GE Healthcare). Fabs served as the analyte and were used with two-fold serial dilutions. Association and dissociation times were monitored at 20°C with a 25 µL/min flowrate, and the binding response was corrected through double referencing. Traces were fit using Scrubber software. Differential scanning fluorimetry The melting temperatures of the Fabs were determined with SYPRO Orange dye at excitation/emission at 490/575 nm. Thermal melts were performed over a temperature range of 25–95 °C at a rate of 0.5 °C per 30 s interval. Fab concentrations of ~5–10 µM were used. Fab complex crystallization and structure determination Fab12E-Asf1 crystal structure (5EII) The Fab12E-Asf1 complex was concentrated to ~10 mg/ml and crystallized in 20% PEG3350, 0.2 M ammonium acetate. Data was collected at GM/CA-CAT beamline 23ID-D. Data were processed using HKL2000 and the structure was solved using Fab structure (3PGF) and yeast Asf1 structure (1ROC). Model building and refinement were performed using Coot [ 36 ] and Phenix [ 37 ], respectively. Fab12E(FNQIKG)-Asf1 structure (6AZ2) Protein complex was crystallized in 20% PEG3350, 0.2 M ammonium chloride. Data were collected at GM/CA-CAT beamline 23ID-D. Data were processed using HKL3000 and the structure was solved using Fab structure (3PGF) and yeast Asf1 structure (1ROC). Model building and refinement were performed using Coot [ 36 ] and Phenix [ 37 ], respectively. Fab12E(FNQIKP)-Asf1 structure (5UCB) The protein preparation and crystallization conditions were identical to the Fab12E-H12-Asf1 structure complex. Data were collected on SBC beamline 19ID and processed using HKL3000. The structure was solved by molecular replacement using the Fab12E-H12-Asf1 complex as the starting model. Model building and refinement were performed using Coot [ 36 ] and Phenix [ 37 ], respectively. Fab1H-Asf1 structure (4RRP) The structure of the Fab1H-Asf1 complex was solved by the Northeastern Structural Genomics group and the details will be described elsewhere. Fab1H(FNQIKG)-Asf1 structure (6AZ2) The Fab12E-Asf1 complex was concentrated to ~8 mg/ml and crystallized in 20% PEG3350, 0.2 M ammonium acetate. Data were collected at GM/CA-CAT beamline 23ID-B. Data were processed using HKL3000 and the structure was solved using Fab structure (3PGF) and yeast Asf1 structure (1ROC). Model building and refinement were performed using Coot [ 36 ] and Phenix [ 37 ], respectively. Fab1H(VSRRLP)-Asf1 structure (5UEA) The protein preparation and crystallization conditions were identical to the Fab12E-H12-Asf1 structure complex. Data were collected on GM/CA-CAT beamline 23ID-D and processed using HKL3000. Data were processed using HKL3000 and the structure was solved using Fab structure (3PGF) and yeast Asf1 structure (1ROC). Model building and refinement were performed using Coot [ 36 ] and Phenix [ 37 ], respectively. IDE-Fab (IDE) complex (5CJO) IDE-Fab (IDE) complex was crystallized in 6% v/v Tacsimate pH 7.0, 0.1 M HEPES pH 7.0, 8% w/v Polyethylene glycol monomethyl ether 5,000, and 8% v/v tert -butanol at 18°C by hanging drop vapor diffusion. Diffraction data were collected at 100K on the SBC 19-ID beamline at Argonne National Laboratory. Data sets were processed using HKL3000, and the structure of IDE-Fab (IDE) complex was determined by molecular replacement, using the cysteine-free IDE structure (4NXO) and the Fab structure in (4IOF) as search models, and Model building and refinement were performed by using PHENIX, and Coot [ 36 ]. The final model (pdb=5CJO) has R work =19.6% and R free =24.5%. Single-particle cryo-EM analysis of Full length, wild-type KcsA Protein was expressed and purified using standard protocols. In brief, pET28a-KcsA [ 38 ] was expressed in E. coli DE3 pLysS (Novagen Millipore), and purified in HEPES, KCl and DDM by cobalt metal affinity chromatography and size exclusion chromatography (SEC). Purified Fab4 elbow variant were added in excess to purified KcsA, and the stoichiometric complex was further purified on SEC. The peak corresponding to the complex was collected, and used directly for grid preparation without further concentration. C-flat 1.2/1.3, 200 Mesh cryoEM grids (Protochips) were plasma cleaned for 30 sec with an air mixture in a Gatan Solarus plasma cleaner. Purified complex at ~3mg/ml in HEPES pH7.4, 200mM KCl, 0.4mM DDM was plunge-frozen using a FEI Vitrobot, operated at 100% humidity, 22°C, blot force of 3, and blot time of 3 sec. Grids were imaged on an FEI Talos 200kV microscope equipped with a Falcon II detector. Data were collected semi-automatically using Serial EM [ 39 ], with a pixel size of 1.936 Å, and a total dose of 68 e − /Å 2 fractionated across 40 frames. Whole-frame drift correction was performed using MotionCor2 [ 40 ], and data were further processed using Eman2 [ 41 ]. Approximately 6000 particles were manually picked for KcsA-Fab4 elbow variant, CTF corrected and classified in 2D. The best 1500 “top view” particles were selected and averaged.

Supplementary Material supplement