Computational protocol: Macromolecular structure phasing by neutron anomalous diffraction

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Protocol publication

[…] Verification of the 113Cd-substituted protein structure was carried out by X-ray crystal diffraction and X-ray fluorescence prior to the large crystal growth. X-ray data collection was performed at 295 K on ESRF (Grenoble, France) beamline ID23-1 using a very heavily attenuated X-ray beam with a quartz capillary encapsulated crystal. The crystallographic data are summarised in . Data integration was carried out using XDS. The resulting intensities were merged and scaled with SCALA. Molecular replacement was performed using MOLREP/CCP4 using an X-ray structure of the oxidised form of Fe-Pf D-rubredoxin at 295 K (pdb code 4AR5) as the probe. Refinement was performed using data to 1.02 Å resolution with automatic water placement using PHENIX.REFINE in conjunction with COOT. All atoms, including water oxygens but with the exception of deuterium atoms, were refined with anisotropic atomic displacements. The final model is accessible under PDB code 5AI3. [...] Using instrument D19 at the ILL (Grenoble, France), a complete and highly redundant neutron dataset was recorded using omega step-scans of 0.07° with an exposure time of 50 seconds per image at a neutron wavelength of 1.17 Å for the majority of the images. The merged data contains a 99.9% complete set of reflections with high redundancy to 1.75 Å resolution. The dataset was processed with the ILL program RETREAT and corrected for effective neutron attenuation using PLATON with a μ value of 1.54 cm−1 at 1.17 Å wavelength (μ = (∑ (coherent absorption cross section) + ∑ (incoherent absorption cross section)) * n atoms/cell volume). The neutron cross section of 113Cd used was 19800 (+/−400) barn for 1.17 Å wavelength. The reflection data from RETREAT was converted into mtz format with POINTLESS using unit cell parameters refined from the X-ray data (a = 34.44 Å; b = 35.14 Å; c = 43.78 Å, α = β = γ = 90°; P212121). Anomalous pairs were handled separately whilst merging data using SCALA up to a resolution of 1.75 Å. The program MTZ2SCA was used to convert the neutron anomalous data mtz file into sca file format for the purpose of compatibility with HKL2MAP and SHELXC/D/E. Data corresponding to a resolution of better than 2.3 Å resolution were excluded from SHELXD for single anomalous dispersion (SAD) calculations. For convenience, SHELXD was directed to search for a single atom of Cd using 10,000 trials and found one position. Density modification was not performed in SHELXE and the resolution was extrapolated to 1.5 Å. The neutron scattering maps calculated using the non-solvent modified experimental phases of the original and inverted hands of the Cd substructure used for the SHELXE run showed clear differences with protein structure features readily identifiable in the map corresponding to the phasing using the inverted hand substructure. This phase set was therefore used thereafter. At the end of the SHELXE run, the correlation coefficient of the best partial structure trace against neutron data was 18.7% with a total of 38 alanine residues built. The output file containing the experimental phases from SHELXE was converted into an mtz file format using F2MTZ (script from T. Grüne). A free Rflag set of reflections of 5% was generated with IMPORT/CCP4. This reflection file was used in ARP/wARP. Neutron data collection took 13 days, with subsequent integration, neutron attenuation correction and phasing achieved using routine crystallography software in ~2 days. The ARP/wARP model was enhanced with cycles of manual building with COOT together with neutron data refinement with a modified scattering table version of PHENIX.REFINE for the 113Cd isotope (neutron coherent scattering length density of 113Cd is −8.0 fm) with automatic water placement. Model refinement statistics are included in . The structure has been deposited under PDB accession code 5AI2. […]

Pipeline specifications

Software tools XDS, CCP4, Molrep, PHENIX, Coot, HKL2MAP, SHELX, ARP/wARP
Applications Small-angle scattering, Protein structure analysis
Chemicals Cadmium, Hydrogen, Iron