Computational protocol: Structural Analysis of the C-Terminal Region (Modules 18–20) of Complement Regulator Factor H (FH)

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Protocol publication

[…] Crystals of FH18–20 were grown at 17°C by vapor diffusion from hanging drops. Drops contained 1 µl of protein solution (16.8 mg/ml) in PBS with an equal volume of well solution (0.1 M sodium malonate, pH 4.0, 12% w/v polyethylene glycol 3350). Crystals grew within forty-eight hours. Crystals were flash frozen in liquid nitrogen after successive soakings in cryoprotectant solutions containing 10% and 25% v/v glycerol. Intensity data were collected (ϕ scans were 1° over 180°) to a resolution of 1.8 Å (the edge of the detector) on beamline I03 at the Diamond Light Source (Oxfordshire, UK). Data were indexed with Mosflm , and subsequently merged and scaled with SCALA . [...] A previously elucidated structure of FH19–20 (PDB ID: 3OXU/chain F ) was used as a search model for molecular replacement using the program PHASER . The resulting model underwent ten cycles of restrained refinement using the program REFMAC . The remaining CCP module (FH18) was built using the PHENIX Autobuild Wizard and the program COOT . This model was subjected to further cycles of restrained refinement and, when appropriate, ligands and water molecules were added to the model using COOT. Disordered regions were carefully modeled into Fo−Fc electron density and changes in R/Rfree (%) values were used to assess final model quality.The final structure was composed of one FH18–20 molecule comprising 185 residues (Gly1045-Lys1230), 17 of which exhibit alternate conformations, 170 water molecules, four glycerol molecules and a phosphate ion. No clear electron density was observed for the first or last residues in the recombinant FH18–20 sequence (Ala1044 or Arg1231), while the Thr1184–K1188 region within CCP 20 was disordered; this region was modeled using the NMR-derived FH19–20 structure (PDB ID: 2BZM ). The R/Rfree values converged for twenty cycles of REFMAC at 18.2% and 22.6%, respectively. Data-reduction and refinement statistics are summarized in . Figures were generated using the PyMOL Molecular Graphics System (Version 1.3, Schrödinger, LLC). [...] The geometry of the model was assessed using MolProbity . Atomic coordinates and the experimental structure factors for our 1.8 Å structure of FH18–20 have been deposited in the Protein Data Bank with the accession code 3SW0 (PDB ID: 3SW0). [...] Synchrotron radiation X-ray scattering data were collected on the X33 beam line of the EMBL (DESY, Hamburg, Germany) using a Pilatus one-megapixel array detector (Dectris, Switzerland) and eight frames of 15-second exposure times. Solutions of FH18–20 and FH19–20 were measured at 20°C in PBS, pH 7.4, 1 mM DL-Dithiothreitol (DTT) at protein concentrations of 1.9, 3.8 and 7.6 mg/ml (for FH18–20) and 2.4, 4.6 and 7.9 mg/ml (for FH19–20). The sample-to-detector distance was 2.7 m, covering a range of momentum transfer 0.07 nm−1CRYSOL . CRYSOL calculates the partial scattering amplitudes of proteins from their atomic coordinates, taking into account the hydration layer and excluded solvent volume. Low resolution shape envelopes were determined from the solution scattering data using the program DAMMIF and the most typical model from multiple reconstructions (10) identified using DAMAVER . Resulting bead models were converted to meshed envelopes and visualized using PYMOL (Version 1.3, Schrödinger, LLC). Superposition of available bead models on three-dimensional structures of FH18–20 or FH19–20, as appropriate, were carried out using the program SUPCOMB13 . Rigid body modeling using the program CORAL (Complexes with Random Loops) was also conducted using the FH18–20 crystal structure, constraining either FH modules 18 and 19, or 19 and 20 as fixed, and refining the relative position and orientation of modules 20 or 18, respectively, against the SAXS data .Analysis of inter-domain flexibility in FH18–20 employed the ensemble optimization method (EOM) . This uses a genetic algorithm to select, from a pool of randomly generated models, an ensemble of possible conformations whose combined theoretical scattering profiles best fit the experimental data. The CCP modules of FH18–20 were treated as rigid bodies and the linkers between them represented as flexible chains of dummy residues. For the pool, 10,000 models were generated from the input structures. A final ensemble of 20 conformations was selected by genetic algorithm after 50 cycles.The discrepancies (χ) between models/ensembles and the experimental data from CRYSOL, DAMMIF, CORAL and EOM are summarised in . This discrepancy is defined as:(1)where N is the number of experimental points, Iexp(sj) and Icalc(sj) are the experimental and calculated scattering intensities, c is a scaling factor and σ(sj) is the experimental error at the momentum transfer sj. […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, PyMOL, MolProbity, CRYSOL, DAMMIF, ATSAS, CORAL, EOM
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens