Research ArticleSTRUCTURAL BIOLOGY

Native phasing of x-ray free-electron laser data for a G protein–coupled receptor

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Science Advances  23 Sep 2016:
Vol. 2, no. 9, e1600292
DOI: 10.1126/sciadv.1600292
  • Fig. 1 Sulfur peaks in the anomalous difference A2AAR Fourier map.

    Sulfur density is contoured at 3 σ and overlaid on the A2AAR crystal structure. Twenty sulfur atoms could be identified from the map. BRIL fusion moiety containing one ordered sulfur atom (M1033) is not shown. Three sulfurs (M-24, C-13, and M1058) are disordered and do not have electron density.

  • Fig. 2 Improvements in electron density at different stages of the phasing process.

    (A) Phaser EP map. (B) Resolve density modified map. (C) Autobuild autotraced map. Omit electron density around the ligand is shown on the top panels. 2mFo-DFc electron density map for helix III is shown on the bottom panels. All maps are contoured at 1.0 σ.

  • Fig. 3 Comparison of resolved water molecules between the room temperature XFEL structure (A2A_S-SAD_1.9) and the cryocooled synchrotron structure (PDB: 4EIY).

    (A) Cartoon representation of the XFEL structure with overlaid waters. Water molecules from the XFEL structure are shown as semitransparent spheres, whereas waters from PDB: 4EYI are shown as dots, colored by location: green, close proximity to ligand (<5 Å); red, sodium ion pocket (<10 Å); cyan, other regions. (B) Conservation of the water positions between PDB: 4EIY and XFEL structures. For each water molecule in PDB: 4EIY, the distance to the closest water in the XFEL structure is shown on the y axis, whereas its B factor is shown on the x axis. Data points are colored the same way as in (A). Positions of water molecules can be considered as conserved if the distance between corresponding water molecules in two structures is less than 1 Å.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1600292/DC1

    fig. S1. A2A-BRIL/ZM241385 microcrystals used for data collection.

    fig. S2. Strength of anomalous signal and sulfur atom search.

    fig. S3. Parameter-space screening results for S-SAD phasing using the X2DF pipeline.

    fig. S4. Effect of different data processing methods on data merging metrics.

    fig. S5. Dependence of anomalous signal measurability on the number of indexed patterns.

    fig. S6. Final 1.9 Å XFEL room temperature A2AAR-BRIL structure (A2A_S-SAD_1.9).

    fig. S7. Structure-factor amplitude difference Fourier map between A2A_S-SAD_2.5 and A2A_MR_2.5 structures.

    fig. S8. B factor comparison between A2A_S-SAD_2.5 and A2A_MR_2.5 structures.

    fig. S9. Comparison of 2mFo-DFc electron density maps for the ligand- and sodium-binding pockets obtained by S-SAD and MR phasing.

    fig. S10. Cα-Cα difference distance matrix between A2A_S-SAD_1.9 and previously determined A2AAR structure (PDB: 4EIY).

    fig. S11. B factor comparison between room temperature A2A_S-SAD_1.9 and previously determined cryocooled A2AAR structure (PDB: 4EIY).

    fig. S12. Distribution of Cys and Met residues in human proteins.

    table S1. Data collection statistics.

    table S2. Data refinement statistics.

    table S3. Comparison of interactions involving charged residues between PDB: 4EIY and A2A_S-SAD_1.9 structures.

    table S4. Comparison of protein and data collection parameters for successful S-SAD phasing of XFEL data.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. A2A-BRIL/ZM241385 microcrystals used for data collection.
    • fig. S2. Strength of anomalous signal and sulfur atom search.
    • fig. S3. Parameter-space screening results for S-SAD phasing using the X2DF pipeline.
    • fig. S4. Effect of different data processing methods on data merging metrics.
    • fig. S5. Dependence of anomalous signal measurability on the number of indexed patterns.
    • fig. S6. Final 1.9 Å XFEL room temperature A2AAR-BRIL structure (A2A_S-SAD_1.9).
    • fig. S7. Structure-factor amplitude difference Fourier map between A2A_S-SAD_2.5 and A2A_MR_2.5 structures.
    • fig. S8. B factor comparison between A2A_S-SAD_2.5 and A2A_MR_2.5 structures.
    • fig. S9. Comparison of 2mFo-DFc electron density maps for the ligand- and sodium-binding pockets obtained by S-SAD and MR phasing.
    • fig. S10. Cα-Cα difference distance matrix between A2A_S-SAD_1.9 and previously determined A2AAR structure (PDB: 4EIY).
    • fig. S11. B factor comparison between room temperature A2A_S-SAD_1.9 and previously determined cryocooled A2AAR structure (PDB: 4EIY).
    • fig. S12. Distribution of Cys and Met residues in human proteins.
    • table S1. Data collection statistics.
    • table S2. Data refinement statistics.
    • table S3. Comparison of interactions involving charged residues between PDB: 4EIY and A2A_SSAD_1.9 structures.
    • table S4. Comparison of protein and data collection parameters for successful S-SAD phasing of XFEL data.

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