Characterisation of recombinant CD64.

(A) The elution of CD64 from an IgG sepharose affinity column (mAU, milli-absorbance units) (B) SDS-PAGE analyses of CD64. Lane 2 represents the non-reduced (NR) recombinant CD64 and Lane 3 represents reduced CD64 (R). The original SDS-PAGE image is shown in S1 Fig. Most of the CD64 was monomeric (arrowed as M) while small amounts of dimer CD64 are arrowed as D. (C) The LC-MS mass spectrum for recombinant CD64 is shown as a single peak at 32.00 kDa at a voltage of 350 V, together with a small peak at 64.01 kDa. (D) The theoretical charge states generated using MassHunter software are labelled. The three strongest peaks are expanded into the panel for clarity.</p

The original SDS-PAGE image used for Fig 2B in the main text is shown.

Lanes 8, 9 and 10 (protein markers, non-reduced CD64 and reduced CD64) were used in Fig 2B. (JPG)</p

The supporting information zip file provides the 100 best-fit models for CD64 corresponding to the fit searches of the X-ray curve at 0.11 mg/ml.

The supporting information zip file provides the 100 best-fit models for CD64 corresponding to the fit searches of the X-ray curve at 0.11 mg/ml.</p

Comparison of the crystal structures of CD64 with density plots of the 100 best-fit models of CD64.

(A-D) Ribbon diagrams of the four CD64 crystal structures correspond to unbound CD64 (PDB code 3RJD) and the three CD64-Fc co-crystals (PDB codes B, 4W4O, C, 4X4M and D, 4ZNE). The glycans in the crystal structures are shown as yellow sticks. (E-H) The density plots of the four sets of putative 100 best-fit CD64 models at four concentrations are shown. The energy-minimised starting structure of CD64 is shown as a black cartoon in the same views in all four panels. The 100 best-fit models were superimposed on the D2 domain. The volumes occupied by the D1, D2 and D3 domains in the 100 best-fit structures are represented as blue, orange and red wireframes respectively in each density plot. Two different views rotated by 90° of each density plot are displayed. In panel E, we also show a stick representation of 10 of the 100 best-fit CD64 models in a smaller scale to clarify how the density plots related to the atomistic models, with the best fit model shown as a blue ribbon.</p

Structural modelling of the SAXS and AUC data for CD64.

Structural modelling of the SAXS and AUC data for CD64.</p

Experimental and modelled Kratky plots for CD64.

The normalised dimensionless Kratky plots for 0.11, 0.22, 0.33 and 0.44 mg/ml CD64 are shown as white, grey, dark grey and black circles respectively. The Kratky plots for each of the modelled X-ray curves for each CD64 concentration are shown as dotted, short dash, long dash and solid red lines respectively. The blue vertical line represents the point of inflection at a Q.RG value of 1.7.</p

Atomistic modelling of the CD64 solution structure.

The 279,162 goodness-of-fit R-factors (○) at four concentrations of CD64 at (A) 0.11 mg/ml, (B) 0.22 mg/ml, (C) 0.33 mg/ml and (D) 0.44 mg/ml were compared with the calculated X-ray RG values for the CD64 models. The R-factors of the 100 best-fit models for each concentration of CD64 are shown as green circles. The experimental Guinier RG values (Fig 4C) are shown as thick vertical black lines, flanked by vertical dashed lines to denote ±5% of the RG value.</p

X-ray scattering curve fits for the best-fit CD64 models.

The interpolated experimental X-ray scattering curves are indicated by open circles and the scattering curves of the best-fit models are indicated by blue continuous lines. The fits correspond to (A) 0.11 mg/ml, (B) 0.22 mg/ml, (C) 0.33 mg/ml and (D) 0.44 mg/ml of CD64 (Fig 4). The red lines represent the modelled curve for the energy-minimised starting structure. The yellow dashed lines represent the calculated scattering curve from the CD64 crystal structure (chain E, PDB code 4X4M). The insets represent the corresponding modelled P(r) curves (blue) overlaid onto the normalised experimental P(r) curves (black) from Fig 4F.</p

Summary of the modelling simulations for CD64.

Details of the thirteen CD64 simulations using the torsion angle Monte Carlo module in SASSIE-web are shown.</p

Experimental and modelled sedimentation analyses of CD64.

(A-D) Sedimentation velocity analyses for CD64 in 30 mM Tris, 150 mM NaCl, pH 7.6, 20°C are shown for A,B, absorbance optics and C,D, interference optics. Up to 30 sedimentation boundaries are shown from a total of 150 scans. The boundary fits are shown in blue (left), together with the corresponding c(s) plot (right). The vertical dashed line (red) shows the average sedimentation coefficient. (E) The concentration dependence of the experimental s20,w values were recorded in the above buffer (■) and also with the addition of 100 mM imidazole to this buffer (●). The solid best-fit line shows the experimental s20,w values. The s020,w values from HYDROPRO are displayed as a dashed line for four known crystal structures (Methods). The s20,w regression line is shown from the scattering best-fit models of CD64 (☆) at four concentrations.</p