Research ArticleBIOCHEMISTRY

Fast iodide-SAD phasing for high-throughput membrane protein structure determination

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Science Advances  12 May 2017:
Vol. 3, no. 5, e1602952
DOI: 10.1126/sciadv.1602952
  • Fig. 1 I-SAD phasing of MP crystal structures.

    Heavy atom substructure determination (SHELXD, left) and phase calculation and extension (SHELXE, right) for each of the four targets investigated. In all four cases, plots of CCall versus CCweak (1000 trials) from SHELXD show a bimodal distribution indicative of a correct iodide ion substructure solution, whereas the difference in contrast (SHELXE) between the two possible hands (red, original; blue, inverted) indicates successful structure solution.

  • Fig. 2 I-SIRAS phasing of MP crystal structures.

    Heavy atom substructure determination (SHELXD, left) and phase calculation and extension (SHELXE, right) for I-SIRAS phasing for each of the four targets investigated. As might be expected, I-SIRAS produces both clearer substructure solutions and better contrast in electron density maps phased using the two possible hands (see Fig. 1 for a comparison).

  • Fig. 3 I-SAD/I-SIRAS phasing of the crystal structure of KR2 using diffraction data collected using serial methods.

    Left: Photograph of crystals, 10 to 15 μm in maximum dimensions, of KR2 in its monomeric blue form. Top right: Heavy atom substructure determination (SHELXD, left) and phase calculation and extension (SHELXE, right) for I-SAD phasing using data collected via serial methods. Bottom right: Heavy atom substructure determination (SHELXD, left) and phase calculation and extension (SHELXE, right) for I-SIRAS phasing using data collected via serial methods.

  • Fig. 4 Cartoon representations of the crystal structures of the four classes of MP solved using the I-SAD/I-SIRAS technique.

    (A) KR2. (B) MACR (noncrystallographic dimer). (C) A2AAR-BRIL-ΔC. (D) NarQ. On the left of each representation, peaks in I-SAD anomalous difference Fourier maps [purple chicken wire, highest-resolution I-SAD data sets for each target (Table 1), contoured at the 3.5 × r.m.s. (root mean square) level] are shown superposed on the Cα backbone of the protein. On the right of each panel, the blue and red lines represent outer and inner lipidic membrane surfaces, respectively, with the hydrophobic region of the lipidic membrane represented in yellow. Iodide ions are shown as orange spheres, divided into three sizes based on the height of anomalous difference map peaks. Two iodide binding sites per crystal structure are highlighted to illustrate their environment. In (D), it is clear that protein residues may change their side-chain conformation upon binding of iodide (conformation in native structures shown as shadows).

  • Fig. 5 Comparison of peaks in anomalous difference Fourier maps in I-SAD and Br-SAD experiments.

    (A) Peaks in anomalous difference Fourier maps in Br-SAD (left) and I-SAD (middle) experiments for NarQ, with a superposition of the two maps shown on the right. (B) Peaks in anomalous difference Fourier maps in Br-SAD (left) and I-SAD (middle) experiments for KR2, with a superposition of the two maps shown on the right. Only the regions close to the proteins are shown, and the maps are contoured at the 3.5 × r.m.s. level. For both NarQ and KR2, the anomalous difference Fourier maps shown are those calculated using anomalous differences in the individual I-SAD data sets of the highest resolution, as detailed in Table 1, and in the merged Br-SAD data sets, as detailed in Table 5.

  • Fig. 6 Comparison of anomalous signal in I-SAD and Br-SAD diffraction data sets for NarQ (A) and KR2 (B).

    (A) Comparisons of SigAno (left) and CCanom (right) in I-SAD (top panel) and Br-SAD (bottom) panel data sets measured from derivatized NarQ crystals. (B) Comparisons of SigAno (left) and CCanom (right) in I-SAD (top panel) and Br-SAD (bottom) panel data sets measured from KR2 crystals. Individual and merged data sets for I-SAD experiments are as detailed in Table 1, whereas those for Br-SAD are as detailed in Table 5.

  • Table 1 I-SAD/I-SIRAS data collection statistics.

    Entries in parentheses represent values for the highest-resolution bin. All statistics and data quality indicators are calculated, treating Friedel’s pairs as separate reflections. Linear merging R value Embedded Image; redundancy-independent merging R value Embedded Image; CC1/2—correlation coefficient between intensities from random half–data sets; Embedded Image.

    Protein/
    space
    group
    Crystal/
    data
    set
    Unit cell dimensions (Å, o)Resolution
    range (Å)
    MultiplicityCompleteness
    (%)
    Rmerge
    (%)
    Rmeas
    (%)
    <I/σ(I)>CC1/2
    (%)
    SigAnoCCanom
    (%)
    SigAnoinnerCCanom_inner
    (%)
    abcαβγ
    NarQ
    F222
    Native57.5873.87235.5690909060–2.0
    (2.1–2.0)
    3.61 (3.50)99.8 (99.7)8.8
    (79.7)
    10.4
    (94.2)
    10.58
    (1.58)
    99.9
    (62.3)
    0.76
    (0.73)
    −3 (−4)0.68−4
    157.2773.64236.1590909060−2.9
    (3.1−2.9)
    6.69 (5.99)99.7 (99.1)15.8
    (77.1)
    17.2
    (84.6)
    10.23
    (2.45)
    99.8
    (87.5)
    1.24
    (0.88)
    47 (26)4.1292
    257.4073.79237.0290909060−3.3
    (3.5−3.3)
    6.73 (6.78)99.7 (99.8)20.8
    (75.9)
    22.6
    (82.2)
    7.33
    (2.36)
    99.6
    (86.1)
    1.15
    (0.76)
    41 (8)3.1790
    357.2273.59236.4290909060−3.1
    (3.3−3.1)
    6.74 (6.53)99.7 (99.7)14.2
    (62.0)
    15.4
    (67.5)
    9.87
    (3.20)
    99.8
    (88.8)
    1.43
    (1.08)
    57 (32)4.0393
    456.9273.20236.2590909060−2.9
    (3.1−2.9)
    6.69 (6.69)99.7 (99.8)11.3
    (50.3)
    12.3
    (54.6)
    12.37
    (3.30)
    99.8
    (93.3)
    1.57
    (0.78)
    64 (5)5.8296
    557.2173.52236.5490909060−2.7
    (2.9−2.7)
    6.69 (6.59)99.8 (99.8)11.4
    (73.3)
    12.4
    (79.6)
    11.93
    (2.47)
    99.9
    (85.3)
    1.51
    (0.82)
    61 (16)6.0397
    657.1273.34236.7490909060−2.9
    (3.1−2.9)
    6.72 (6.52)99.4 (99.5)11.3
    (67.4)
    12.3
    (73.2)
    12.20
    (2.67)
    99.9
    (87.1)
    1.53
    (0.80)
    60 (1)5.8596
    756.9973.12236.0790909060−2.9
    (3.1−2.9)
    6.73 (6.68)98.9 (98.4)11.1
    (71.4)
    12.0
    (77.4)
    12.56
    (2.51)
    99.7
    (82.5)
    1.57
    (0.80)
    62 (8)6.0097
    857.2473.66236.4390909060−2.7
    (2.9−2.7)
    6.73 (6.68)99.8 (99.8)12.6
    (71.2)
    13.6
    (77.4)
    11.03
    (2.67)
    99.9
    (86.8)
    1.41
    (0.94)
    54 (16)5.1596
    Merged57.1773.48236.4590909060−2.7
    (2.9−2.7)
    42.95 (13.26)99.9 (99.8)17.5
    (76.0)
    17.7
    (79.1)
    20.68
    (3.12)
    99.8
    (91.7)
    2.26
    (0.85)
    78 (17)10.1498
    KR2
    I222
    Native40.6283.66234.4690909060−2.5
    (2.65−2.5)
    6.58 (6.45)99.7 (99.6)9.6
    (85.9)
    10.5
    (93.7)
    13.73
    (2.10)
    99.8
    (81.2)
    0.84
    (0.81)
    15 (19)1.4244
    140.5083.66234.1390909060−2.8
    (3.0−2.8)
    6.85 (6.72)99.7 (99.8)13.5
    (83.0)
    14.6
    (89.9)
    11.87
    (2.44)
    99.8
    (80.7)
    1.22
    (0.80)
    41 (10)6.7697
    240.5283.63233.8090909060−2.8
    (3.0−2.8)
    6.82 (6.71)99.8 (99.7)13.7
    (77.0)
    14.8
    (83.5)
    11.92
    (2.60)
    99.6
    (87.2)
    1.24
    (0.79)
    44 (7)7.2698
    340.5183.75234.1090909060−2.5
    (2.65−2.5)
    6.73 (6.60)99.9 (100)11.3
    (75.4)
    12.3
    (82.0)
    11.95
    (2.35)
    99.9
    (79.7)
    1.22
    (0.76)
    42 (0)7.6498
    440.7283.99234.2190909060−2.65
    (2.8−2.65)
    6.71 (6.40)99.8 (99.7)10.6
    (64.0)
    11.5
    (69.7)
    12.70
    (2.66)
    99.6
    (89.0)
    1.25
    (0.77)
    46 (5)6.9698
    Merged40.5683.76234.0690909060−2.5
    (2.65−2.5)
    22.06 (6.60)100 (100)13.7
    (75.1)
    14.0
    (81.6)
    19.43
    (2.30)
    99.9
    (80.0)
    1.64
    (0.75)
    60 (1)13.2399
    MACR
    P1
    Native40.7656.8557.6563.679.080.160−2.0
    (2.2−2.0)
    1.05 (1.07)76.2 (73.1)7.3
    (26.7)
    10.3
    (37.9)
    6.43
    (1.81)
    99.5
    (86.2)
    n/an/an/an/a
    140.7756.8957.2863.878.680.260−2.2
    (2.4−2.2)
    1.86 (1.81)70.1 (23.0)5.1
    (27.2)
    7.2
    (38.5)
    10.40
    (2.61)
    99.5
    (87.0)
    1.16
    (0.88)
    43 (8)3.6191
    240.7556.8757.3263.678.480.260−2.4
    (2.6−2.4)
    1.85 (1.79)72.5 (20.0)6.4
    (44.9)
    9.0
    (63.4)
    7.86
    (1.55)
    99.5
    (72.4)
    1.07
    (0.87)
    36 (22)3.1489
    340.7956.7757.2963.678.580.260−2.0
    (2.2−2.0)
    1.88 (1.84)81.9 (72.3)5.1
    (38.3)
    7.2
    (54.2)
    8.64
    (1.86)
    99.7
    (70.4)
    1.07
    (0.76)
    35 (−5)3.4990
    Merged40.7756.8457.3063.778.580.260−2.0
    (2.2−2.0)
    3.68 (1.84)89.4 (71.8)7.9
    (38.7)
    9.0
    (54.8)
    11.03
    (1.81)
    99.7
    (69.5)
    1.22
    (0.75)
    46 (−5)4.8595
    A2AAR-BRIL-ΔC
    C2221
    Native39.62179.54139.7190909060−2.4
    (2.6−2.4)
    2.94 (2.98)99.6 (99.8)9.4
    (41.1)
    11.5
    (50.2)
    9.60
    (2.74)
    99.5
    (80.4)
    0.77
    (0.77)
    −4 (−1)0.74−14
    139.63179.66139.5790909060−3.4
    (3.6−3.4)
    6.93 (6.96)100 (100)24.8
    (73.6)
    26.8
    (79.5)
    7.33
    (2.56)
    99.1
    (81.2)
    0.96
    (0.79)
    17 (0)1.9471
    239.65179.79139.8190909060−2.8
    (3.0−2.8)
    6.87 (6.87)99.7 (99.0)18.5
    (78.1)
    20.0
    (84.5)
    8.83
    (2.29)
    99.4
    (76.0)
    0.98
    (0.76)
    18 (−5)2.5782
    339.40179.87139.5290909060−3.6
    (3.8−3.6)
    6.67 (6.54)99.9 (99.8)23.5
    (72.1)
    25.5
    (78.5)
    8.45
    (3.21)
    99.2
    (82.8)
    1.13
    (1.08)
    46 (41)1.9672
    439.57179.76139.9990909060−3.8
    (4.0−3.8)
    6.83 (6.79)99.9 (99.9)30.7
    (84.7)
    33.3
    (91.9)
    6.67
    (2.97)
    98.6
    (72.0)
    0.96
    (0.91)
    19 (8)1.5963
    539.60179.10139.4290909060−3.0
    (3.2−3.0)
    3.02 (2.98)98.9 (99.0)14.5
    (65.7)
    17.6
    (80.1)
    7.06
    (1.67)
    98.7
    (56.6)
    0.93
    (0.75)
    16 (−3)2.0674
    639.50179.46139.4090909060−3.4
    (3.6−3.4)
    6.85 (6.52)99.9 (100)19.9
    (69.1)
    21.5
    (75.2)
    9.61
    (3.20)
    99.4
    (80.6)
    1.07
    (0.89)
    27 (5)2.4681
    739.63179.65139.9690909060−3.2
    (3.4−3.2)
    6.27 (5.83)99.9 (99.7)21.1
    (68.3)
    23.1
    (75.1)
    8.86
    (2.61)
    98.9
    (84.4)
    1.02
    (0.92)
    24 (12)2.0676
    Merged39.57179.61139.6790909060−2.8
    (3.0−2.8)
    27.10 (6.88)99.8 (99.1)24.5
    (77.6)
    24.9
    (83.9)
    14.55
    (2.25)
    99.8
    (76.1)
    1.24
    (0.73)
    35 (−3)4.8394
  • Table 2 I-SAD structure solution and refinement statistics.
    Protein
    NarQKR2MACRA2AAR-BRIL-ΔC
    Structure solution and initial model building
      DataEight anomalous +
    native
    Data set #8 +
    native
    Four anomalous +
    native
    Three anomalous +
    native
    Seven anomalous +
    native
      MethodI-SADI-SIRASI-SADI-SIRASI-SADI-SIRASI-SADI-SIRAS
      Initial resolution range (Å)60−2.760−2.760−2.560−2.560−2.060−2.060−2.860−2.8
      Number of residues in
    the protein
    237288220447
      Substructure14·I10·I16·I17·I26·I
    14·I
    16·I8·I10·I
      Resolution cutoff for
    substructure search (Å)
    3.02.82.82.82.52.93.73.9
      Resolution extension by
    native data (Å)
    1.951.952.52.52.02.02.42.4
      CCall/CCweak40/15.431.8/18.142.6/20.737.7/25.128.8/15.329.8/19.635.0/14.836.7/21.4
      Solvent content used
    in SHELXE (%)
    4747585950504949
      CCmap, highest-resolution shell (%)7477678487817374
      Number of Ala residues
    traced by SHELXE
    178187212208190272155250
      Rwork/Rfree after initial
    model building (%)
    23.0/28.5*24.8/32.9*22.4/30.1*22.6/29.3*24.2/29.2†‡ 29.4/32.729.7/46.2* 29.9/32.730.7/32.929.5/45.8*
      Number of residues built
    in initial model building
    215*216*235*265*343†‡ 30069* 319277168*
    Structure refinement
      Data set (see Table 1)8332
      Rwork/Rfree final (%)19.0/26.219.0/22.617.7/21.923.4/29.4
      Number in the ASU (asymmetric unit)
        Protein residues223268431388
        Water molecules60497864
        Iodide ions1220226
      Averaged B-factors (Å2)
        Protein residues43403838
        Water molecules34444727
        Iodide ions70667150
      Ramachandran plot
        Preferred216 (99.1%)259 (97.4%)419 (98.4%)373 (97.9%)
        Allowed2 (0.9%)6 (2.2%)7 (1.6%)7 (1.8%)
        Outliers01 (0.4%)01 (0.3%)

    *Initial model built by ARP-wARP.

    †Phasing carried out using phenix.autosol (all others used SHELXC/D/E).

    ‡Initial model built by phenix.autobuild.

    • Table 3 Data collection, structure solution, and initial model building statistics resulting from I-SAD/I-SIRAS phasing of the crystal structure of KR2 using diffraction data collected using serial methods.
      I-SADI-SIRASI-SADI-SIRAS
      Data
      collection
      No. of crystals13639Structure
      solution
      and Refinement
      Initial resolution
      range (Å)
      60–2.860–2.9
      MethodSADSIRASNumber of
      residues in
      the protein
      288288
      Space groupI222I222Substructure16·I12·I
      Unit cella40.641.0Resolution
      cutoff for
      substructure
      search (Å)
      3.93.4
      b83.684.0Resolution
      extension
      by native
      data (Å)
      2.52.5
      c233.5234.3CCall/CCweak38.7/11.026.7/9.6
      No. of frames13,6003900Solvent
      content (%)
      6360
      Oscillation
      range (°)
      0.10.1CCmap,
      highest-resolution
      shell (%)
      4767
      Resolution
      range (Å)
      60–2.8 (3.0–2.8)60–2.9 (3.1–2.9)Number of
      Ala
      residues
      traced by
      SHELXE
      122165
      Measured
      reflections
      469,213 (85,461)124,613 (22,610)Rwork/Rfree
      after initial
      model
      building (%)
      29.6/35.724.2/31.6*
      29.9/35.4
      Multiplicity24.79 (24.13)7.19 (7.13)Number of
      residues
      built in
      initial model
      building
      207248*
      196
      Completeness (%)100 (100)99.8 (100)
      Rmerge (%)61.7 (220.7)23.8 (154.6)
      Rmeas (%)63.0 (225.4)25.6 (166.6)
      <I/σ(I)>10.64 (2.27)7.71 (1.69)
      CC1/2 (%)99.8 (70.1)99.3 (56.2)
      SigAno1.21 (0.78)1.03 (0.73)
      CCanom (%)39 (0)29 (−1)

      *Model built by ARP-wARP.

      †Model built by phenix.autobuild.

      • Table 4 The analysis of presence at the membrane hydrophobic-hydrophilic interface of arginine (R), lysine (K), histidine (H), tryptophan (W), and tyrosine (Y).

        As outlined in the main text, 445 unique transmembrane protein crystal structures deposited in the PDB were analyzed with, in each case, the membrane surfaces represented by two parallel planes calculated by Lomize et al. (42). Amino acid residues were considered to be present at the membrane interfaces if its Cα atom was not more than 7 Å from the membrane surfaces defined above.

        R/K/HR/KW/YRKHWY
        Number of entries where the amino acid
        residues are present at the membrane interface
        445
        (100%)
        442
        (99.3%)
        428
        (96.2%)
        429
        (96.4%)
        414
        (93.0%)
        353
        (79.3%)
        393
        (88.3%)
        422
        (94.8%)
      • Table 5 Br-SAD data collection statistics.

        Entries in parentheses represent values for the highest-resolution bin. All statistics and data quality indicators are calculated treating Friedel’s pairs as separate reflections. Linear merging R value Embedded Image; redundancy-independent merging R value Embedded Image; CC1/2—correlation coefficient between intensities from random half–data sets; Embedded Image.

        Protein/
        space
        group
        Crystal/
        data
        set
        λ (Å)Unit cell dimensions (Å, o)
        abcαβγResolution
        (Å)
        MultiplicityCompleteness
        (%)
        Rmerge
        (%)
        Rmeas
        (%)
        <I/σ(I)>CC1/2
        (%)
        SigAnoCCanom
        (%)
        SigAnoinnerCCanom_inner
        (%)
        NarQ
        F222
        10.872957.8774.79237.8590909060–2.8
        (3.0–2.8)
        2.09 (2.11)83.2 (45.2)14.5
        (105.4)
        18.2
        (132.8)
        5.53
        (1.21)
        99.4
        (47.7)
        0.82
        (0.71)
        6 (10)1.1237
        20.872957.7074.34237.7090909060–2.8
        (3.0–2.8)
        4.03 (4.12)98.2 (92.6)18.1
        (106.9)
        20.8
        (122.7)
        6.99
        (1.36)
        99.5
        (58.3)
        0.83
        (0.74)
        8 (3)1.5655
        30.872958.2173.57237.4290909060–2.8
        (3.0–2.8)
        4.81 (4.87)99.2 (99.2)21.3
        (105.1)
        24.0
        (117.9)
        5.85
        (1.43)
        99.5
        (65.2)
        0.82
        (0.67)
        13 (1)1.2745
        140.872957.5373.91237.2390909060–2.6
        (2.8–2.6)
        4.31 (4.06)93.2 (68.4)15.7
        (101.1)
        17.9
        (116.5)
        7.38
        (1.26)
        99.5
        (60.1)
        0.86
        (0.71)
        11 (3)1.5061
        150.872957.5274.44237.7690909060–2.8
        (3.0–2.8)
        2.58 (2.40)93.5 (87.6)14.0
        (59.6)
        17.5
        (74.6)
        5.83
        (1.47)
        98.9
        (66.1)
        0.85
        (0.74)
        18 (−2)1.1456
        160.872957.7674.22238.6890909060–2.6
        (2.8–2.6)
        2.89 (2.98)97.4 (98.4)10.8
        (73.5)
        13.0
        (88.8)
        8.13
        (1.55)
        99.6
        (59.7)
        0.87
        (0.75)
        9 (2)1.8074
        240.872957.5174.20237.2690909060–2.2
        (2.4–2.2)
        2.31 (2.22)91.6 (76.8)11.4
        (125.6)
        14.5
        (160.1)
        5.85
        (0.62)
        99.6
        (69.2)
        0.83
        (0.65)
        12 (4)1.6979
        Merged57.7374.21237.7090909060–2.8
        (3.0–2.8)
        22.3 (21.3)94.2 (100)19.7
        (85.9)
        20.1
        (88.0)
        17.3
        (5.0)
        99.9
        (96.6)
        0.98
        (0.75)
        20 (3)2.7790
        KR2
        I222
        320.9196840.6783.73234.4690909060–2.8
        (3.0–2.8)
        6.68 (6.45)89.4 (44.7)24.0
        (176.7)
        26.1
        (191.6)
        7.44
        (1.11)
        99.6
        (49.4)
        0.97
        (0.65)
        43 (3)1.7567
        330.9196840.7883.62234.3890909060–3.0
        (3.2–3.0)
        6.82 (7.05)100 (100)26.3
        (159.7)
        28.6
        (172.5)
        7.43
        (1.33)
        99.6
        (47.5)
        0.92
        (0.70)
        21 (−8)1.8771
        370.9196840.6783.32234.1090909060–2.8
        (3.0–2.8)
        6.77 (6.64)99.5 (99.2)16.1
        (118.5)
        17.4
        (128.7)
        9.85
        (1.61)
        99.8
        (78.1)
        0.88
        (0.72)
        17 (7)1.6967
        380.9196840.5883.31234.0490909060–2.8
        (3.0–2.8)
        6.85 (7.05)99.6 (99.4)14.4
        (103.8)
        15.6
        (112.3)
        10.06
        (1.73)
        99.8
        (84.9)
        0.90
        (0.76)
        19 (7)1.7569
        390.9196840.5683.46233.4590909060–3.0
        (3.2–3.0)
        6.74 (6.54)96.1 (79.3)17.0
        (121.3)
        18.4
        (131.7)
        9.52
        (1.35)
        99.9
        (74.9)
        0.90
        (0.73)
        21 (14)1.6561
        Merged40.6583.49234.0990909060–2.8
        (3.0–2.8)
        31.56 (23.48)100 (100)23.9
        (166.0)
        24.3
        (169.7)
        16.72
        (2.49)
        99.9
        (85.7)
        1.03
        (0.74)
        23 (0)2.6380

      Supplementary Materials

      • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602952/DC1

        fig. S1. Comparison of peaks in anomalous difference Fourier maps calculated from individual data sets in I-SAD experiment.

        fig. S2. The distribution of positively charged and aromatic residues in the crystal structures obtained in I-SAD experiment.

        fig. S3. The bound iodide ions and their environment.

      • Supplementary Materials

        This PDF file includes:

        • fig. S1. Comparison of peaks in anomalous difference Fourier maps calculated from individual data sets in I-SAD experiment.
        • fig. S2. The distribution of positively charged and aromatic residues in the crystal structures obtained in I-SAD experiment.
        • fig. S3. The bound iodide ions and their environment.

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