Metallohelices that kill Gram-negative pathogens using intracellular antimicrobial peptide pathways
Simpson, Daniel; Hapeshi, Alexia; Rogers, Nicola J.; Brabec, Viktor; Clarkson, Guy J.; Fox, David; Hrabina, Ondrej; Kay, Gemma; King, Andrew; Malina, Jaroslav; Millard, Andrew; Moat, John; Roper, David I.; Song, Hualong; Waterfield, Nick; Scott, Peter; Chemical Science  (2019) –; doi:10.1039/C9SC03532J

data_1552285
_publ_section_title
;
 Metallohelices that kill Gram-negative pathogens using intracellular
 antimicrobial peptide pathways
;
_journal_name_full               'Chemical Science'
_journal_paper_doi               10.1039/C9SC03532J
_journal_year                    2019
_chemical_formula_moiety
'2(C126 H108 N12 O9 Zn2), 6(Cl O4), 2.5(C2 H3 N)'
_chemical_formula_sum            'C257 H223.5 Cl6 N26.5 O42 Zn4'
_chemical_formula_weight         4829.29
_space_group_crystal_system      triclinic
_space_group_IT_number           2
_space_group_name_Hall           '-P 1'
_space_group_name_H-M_alt        'P -1'
_symmetry_space_group_name_Hall  '-P 1'
_symmetry_space_group_name_H-M   'P -1'
_atom_sites_solution_hydrogens   geom
_audit_creation_date             2019-01-06
_audit_creation_method
;
Olex2 1.2
(compiled 2018.05.29 svn.r3510 for OlexSys, GUI svn.r5506)
;
_audit_update_record
;
2019-03-21 deposited with the CCDC.    2019-09-04 downloaded from the CCDC.
;
_cell_angle_alpha                90.746(3)
_cell_angle_beta                 99.035(3)
_cell_angle_gamma                90.078(2)
_cell_formula_units_Z            2
_cell_length_a                   19.4539(5)
_cell_length_b                   23.9678(9)
_cell_length_c                   30.5016(15)
_cell_measurement_reflns_used    6084
_cell_measurement_temperature    100(2)
_cell_measurement_theta_max      59.5520
_cell_measurement_theta_min      2.5310
_cell_volume                     14044.2(9)
_computing_cell_refinement       'CrysAlisPro 1.171.39.46 (Rigaku OD, 2018)'
_computing_data_collection       'CrysAlisPro 1.171.39.46 (Rigaku OD, 2018)'
_computing_data_reduction        'CrysAlisPro 1.171.39.46 (Rigaku OD, 2018)'
_computing_molecular_graphics    'Olex2 (Dolomanov et al., 2009)'
_computing_publication_material  'Olex2 (Dolomanov et al., 2009)'
_computing_structure_refinement  'ShelXL (Sheldrick, 2015)'
_diffrn_ambient_temperature      100(2)
_diffrn_detector                 'CCD plate'
_diffrn_detector_area_resol_mean 10.2833
_diffrn_detector_type            Ruby
_diffrn_measured_fraction_theta_full 0.983
_diffrn_measured_fraction_theta_max 0.983
_diffrn_measurement_details
'List of Runs (angles in degrees, time in seconds): # Type Start End Width t~exp~ \w \q \k \f Frames -------------------------------------------------------------------------- 1 \w -37.00 33.00 1.00 17.50 -- -41.71 38.00 0.00 70 2 \w -120.00 -10.00 1.00 17.50 -- -41.71 -19.00 30.00 110 3 \w -50.00 30.00 1.00 17.50 -- -41.71 38.00-150.00 80 4 \w 55.00 105.00 1.00 17.50 -- 41.71-146.00 135.00 50 5 \w 20.00 86.00 1.00 17.50 -- 41.71-126.00 -57.00 66 6 \w 126.00 178.00 1.00 70.00 -- 114.00 61.00-180.00 52 7 \w 85.00 125.00 1.00 70.00 -- 114.00 61.00-180.00 40 8 \w 57.00 147.00 1.00 70.00 -- 114.00 -77.00-150.00 90 9 \w 132.00 175.00 1.00 70.00 -- 114.00 30.00 -60.00 43 10 \w 39.00 91.00 1.00 70.00 -- 114.00 -30.00 90.00 52 11 \w 86.00 113.00 1.00 70.00 -- 114.00 30.00 -60.00 27 12 \w 57.00 116.00 1.00 70.00 -- 114.00-111.00-180.00 59 13 \w 104.00 178.00 1.00 70.00 -- 114.00 111.00-120.00 74 14 \w 45.00 73.00 1.00 70.00 -- 114.00-111.00 -30.00 28 15 \w 42.00 89.00 1.00 70.00 -- 114.00 -45.00-150.00 47 16 \w 58.00 146.00 1.00 70.00 -- 114.00 178.00 90.00 88 17 \w 68.00 107.00 1.00 70.00 -- 114.00 -94.00 90.00 39 18 \w 38.00 116.00 1.00 70.00 -- 114.00-111.00 60.00 78 19 \w 42.00 98.00 1.00 70.00 -- 114.00 -45.00 150.00 56 20 \w 88.00 178.00 1.00 70.00 -- 114.00 30.00-120.00 90'
_diffrn_measurement_device       'four-circle diffractometer'
_diffrn_measurement_device_type  'Xcalibur, Ruby, Gemini'
_diffrn_measurement_method       '\w scans'
_diffrn_orient_matrix_type
'CrysAlisPro convention (1999,Acta A55,543-557)'
_diffrn_orient_matrix_UB_11      -0.0012374000
_diffrn_orient_matrix_UB_12      0.0604924000
_diffrn_orient_matrix_UB_13      -0.0165650000
_diffrn_orient_matrix_UB_21      0.0134775000
_diffrn_orient_matrix_UB_22      -0.0212114000
_diffrn_orient_matrix_UB_23      -0.0457735000
_diffrn_orient_matrix_UB_31      -0.0790352000
_diffrn_orient_matrix_UB_32      -0.0047892000
_diffrn_orient_matrix_UB_33      -0.0156966000
_diffrn_radiation_monochromator  graphite
_diffrn_radiation_probe          x-ray
_diffrn_radiation_type           CuK\a
_diffrn_radiation_wavelength     1.54178
_diffrn_reflns_av_R_equivalents  0.1177
_diffrn_reflns_av_unetI/netI     0.1930
_diffrn_reflns_Laue_measured_fraction_full 0.983
_diffrn_reflns_Laue_measured_fraction_max 0.983
_diffrn_reflns_limit_h_max       19
_diffrn_reflns_limit_h_min       -23
_diffrn_reflns_limit_k_max       28
_diffrn_reflns_limit_k_min       -28
_diffrn_reflns_limit_l_max       34
_diffrn_reflns_limit_l_min       -36
_diffrn_reflns_number            94201
_diffrn_reflns_point_group_measured_fraction_full 0.983
_diffrn_reflns_point_group_measured_fraction_max 0.983
_diffrn_reflns_theta_full        66.596
_diffrn_reflns_theta_max         66.596
_diffrn_reflns_theta_min         2.340
_diffrn_source                   'fine-focus sealed X-ray tube'
_diffrn_source_type              'Enhance (Cu) X-ray Source'
_exptl_absorpt_coefficient_mu    1.470
_exptl_absorpt_correction_T_max  1.00000
_exptl_absorpt_correction_T_min  0.89963
_exptl_absorpt_correction_type   multi-scan
_exptl_absorpt_process_details
'CrysAlisPro 1.171.39.46 (Rigaku Oxford Diffraction, 2018) Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm.'
_exptl_crystal_colour            colourless
_exptl_crystal_colour_primary    colourless
_exptl_crystal_density_diffrn    1.142
_exptl_crystal_description       block
_exptl_crystal_F_000             5018
_exptl_crystal_size_max          0.2
_exptl_crystal_size_mid          0.12
_exptl_crystal_size_min          0.03
_refine_diff_density_max         1.413
_refine_diff_density_min         -0.574
_refine_diff_density_rms         0.079
_refine_ls_extinction_method     none
_refine_ls_goodness_of_fit_ref   0.927
_refine_ls_hydrogen_treatment    constr
_refine_ls_matrix_type           full
_refine_ls_number_parameters     3166
_refine_ls_number_reflns         48750
_refine_ls_number_restraints     495
_refine_ls_restrained_S_all      0.927
_refine_ls_R_factor_all          0.1901
_refine_ls_R_factor_gt           0.1034
_refine_ls_shift/su_max          0.001
_refine_ls_shift/su_mean         0.000
_refine_ls_structure_factor_coef Fsqd
_refine_ls_weighting_details
'w=1/[\s^2^(Fo^2^)+(0.1054P)^2^] where P=(Fo^2^+2Fc^2^)/3'
_refine_ls_weighting_scheme      calc
_refine_ls_wR_factor_gt          0.2460
_refine_ls_wR_factor_ref         0.3181
_reflns_Friedel_coverage         0.000
_reflns_number_gt                18603
_reflns_number_total             48750
_reflns_threshold_expression     'I > 2\s(I)'
_cod_data_source_file            c9sc03532j3.cif
_cod_data_source_block           ds5x
_cod_original_formula_sum        'C257 H223.50 Cl6 N26.50 O42 Zn4'
_cod_database_code               1552285
_shelx_shelxl_version_number     2018/3
_chemical_oxdiff_formula         'C20 H20 N2 O2 Cl Zn'
_shelx_estimated_absorpt_t_max   0.957
_shelx_estimated_absorpt_t_min   0.758
_reflns_odcompleteness_completeness 89.95
_reflns_odcompleteness_iscentric 1
_reflns_odcompleteness_theta     75.12
_olex2_refinement_description
;
The asymmetric unit contains two crystallographically independent metallohelicies of opposite handedness.
Six perchlorate anions and three acetonitrile molecules were located and refined but the other two 
perchlorates and additional solvent were not located (see below).  
Acetonitrile N900-C902 was refined at half occupancy.

Disorder modeling.
Several of the perchlorates and part of a ligand were modeled as disordered. The occupancy of the 
disordered components was originally assigned to a free variable but once these had settled at the 
later stages of the refinement, the occupancies were fixed for the final refinement cycles.
Perchlorate Cl50 was modeled as disordered over two positions related by a rotation about the O51-Cl50 bond. 
The occupancies were fixed at 50:50
Perchlorate Cl70 was modeled as disordered over two positions related by a rotation about the O71-Cl70 bond. 
The occupancies were fixed at 50:50
Perchlorate Cl60 was modeled over two closely related positions. 
The occupancy was fixed at 70:30 Cl60-O64 to Cl6A to O64A.
The minor component oxygens were refined isotropically.
Phenyl C218-C223 was modeled as disordered over two positions related by a small displacement. 
The disorder was traced back to the benzylic carbon C217.  The occupancy of the two components 
was fixed at 60:40 C217-C223 to C1A-C1G.
Several DFIX, DANG and SIMU restraints were used to give the disordered components reasonably 
bond lengths, angles and thermal parameters

The solvent masking algorithm in Olex2 was used to treat the electron density from those anions and 
solvent molecules that were not located as a diffuse contribution to the overall scattering without 
specific atom positions.

The total solvent accessible volume was 3470 Angstroms cubed (24.7 percent of cell volume). 
The analysis identifies three main voids large enough to hold solvent or counter ions along with 
their postulated contents that could fill the volume and account for the electron density contribution.

These are tabulated below with the following volumes and electron counts and coordinates

Void    Grid points  Vol/A cubed  Percent of cell     electron count        Centre of mass (frac)   
   1       311125     2528.6         18.0               703.2              (-0.639,-0.429,-0.000)  
   2        62453      507.6          3.6               164.9              ( 0.000, 0.500, 0.500)
   3        36197      294.2          2.1               109.0              ( 0.500, 1.000, 0.500)

Volumes of possible solvents/counter ions in voids calculated simply using the vdw radius of each atom type 
Approximate volume of a perchlorate 84 Angstroms cubed
Electron count for ClO4 is 50. 

Approximate volume of acetonitrile 80 Angstroms cubed
Electron count for acetonitrile is 22. 

As six ClO4 were located in the structure, another two perchlorate counter ions are required to balance 
the four Zn ions in the asymmetric unit but the void calculation is for the whole cell so need four ClO4s

The postulated contents of these voids based on the values above is given below

Void 1
Void volume and electron count from Olex2
Volume 2529   electron count 703

Possible Void 1 contents
2 x ClO4 and 27 x acetonitrile
Calculated volume 2328 and electron count 694

Void 1 calculations
Calculated volume (2 ClO4 x 84) + (27 acetonitrile x 80) = 168 + 2160 = 2328 Angstroms cubed

Calculated electron count = (2 ClO4 x 50) + (27 acetonitrile x 22) 
= 100 + 594 = 694 electrons

Void 2 
Void volume and electron count from Olex2
Volume 508 electron count 165

Possible Void 2 contents
1 x ClO4 and 5 x acetonitrile
Calculated volume 484 and electron count 160

Void 2 calculation
Calculated volume (1 ClO4 x 84) + (5 x acetonitrile x 80)= 84 + 400 = 484 Angstroms cubed
Calculated electron count = (1 ClO4 x 50) + (5 acetonitrile x 22) = 50 + 110 = 160 electrons

Void 3
Void volume and electron count from Olex2
Volume 294 electron count 109

Possible Void 3 contents
1 x ClO4 and 2 x acetonitrile
Calculated volume 244 and electron count 94

Void 3 calculation
Calculated volume (1 ClO4 x 84) + (2 acetonitrile x 80)= 84 +160 = 244 Angstroms cubed
Calculated electron count = (1 ClO4 x 50) + (2 acetonitrile x 22) = 50 + 44 = 94 electrons

That is a total of four perchlorate ions and 34 acetonitrile that would account for the calculated 
electron density using the solvent masking algorithm in Olex2

1. Fixed Uiso
 At 1.2 times of:
  All C(H) groups, All C(H,H) groups, All C(H,H,H,H) groups
 At 1.5 times of:
  All C(H,H,H) groups
2. Restrained distances
 Cl70-O72A
 1.4 with sigma of 0.02
 Cl70-O73A
 1.4 with sigma of 0.02
 Cl70-O74A
 1.4 with sigma of 0.02
 Cl60-O61 = Cl60-O62 = Cl60-O63 = Cl60-O64
 1.414 with sigma of 0.02
 O61-O62 = O61-O63 = O61-O64 = O62-O63 = O62-O64 = O63-O64
 2.309 with sigma of 0.04
 O61A-O62A = O61A-O63A = O61A-O64A = O62A-O63A = O62A-O64A = O63A-O64A
 2.309 with sigma of 0.04
3. Uiso/Uaniso restraints and constraints
C50 \\sim C51 \\sim C52 \\sim C53 \\sim C54 \\sim C55: within 1.7A with sigma
of 0.004 and sigma for terminal atoms of 0.008
C184 \\sim C185 \\sim C186 \\sim C193 \\sim C194 \\sim C195: within 1.7A with
sigma of 0.004 and sigma for terminal atoms of 0.008
N17 \\sim C169 \\sim C170 \\sim C171 \\sim C172 \\sim C173: within 1.7A with
sigma of 0.004 and sigma for terminal atoms of 0.008
C241 \\sim C242 \\sim C243 \\sim C244 \\sim C245 \\sim C246: within 1.7A with
sigma of 0.004 and sigma for terminal atoms of 0.008
O11 \\sim C147 \\sim C148 \\sim C149 \\sim C150 \\sim C151 \\sim C152 \\sim
C153: within 1.7A with sigma of 0.004 and sigma for terminal atoms of 0.008
N8 \\sim C80 \\sim C81 \\sim C82 \\sim C83 \\sim C84: within 1.7A with sigma of
0.008 and sigma for terminal atoms of 0.016
O2 \\sim C16 \\sim C21 \\sim C22 \\sim C23 \\sim C24 \\sim C25 \\sim C26 \\sim
C27: within 1.7A with sigma of 0.004 and sigma for terminal atoms of 0.008
C43 \\sim C44 \\sim C45 \\sim C46 \\sim C47: within 1.7A with sigma of 0.004
and sigma for terminal atoms of 0.008
C218 \\sim C219 \\sim C220 \\sim C221 \\sim C222 \\sim C223: within 1.7A with
sigma of 0.004 and sigma for terminal atoms of 0.008
C217 \\sim C218 \\sim C223 \\sim C222 \\sim C221 \\sim C220 \\sim C219: within
2A with sigma of 0.008 and sigma for terminal atoms of 0.016
O53A \\sim O54 \\sim O54A \\sim O53 \\sim O52A \\sim O52 \\sim O51: within 1.7A
with sigma of 0.004 and sigma for terminal atoms of 0.008
C901 \\sim C902 \\sim N900: within 1.7A with sigma of 0.004 and sigma for
terminal atoms of 0.008
N21 \\sim C215 \\sim C214 \\sim C213 \\sim C212 \\sim C211: within 1.7A with
sigma of 0.01 and sigma for terminal atoms of 0.02
C1A \\sim C1B \\sim C1C \\sim C1D \\sim C1E \\sim C1F \\sim C1G: within 1.7A
with sigma of 0.004 and sigma for terminal atoms of 0.008
4. Rigid body (RIGU) restrains
 O62, O64, Cl60, O63, O61
 with sigma for 1-2 distances of 0.002 and sigma for 1-3 distances of 0.002
5. Others
 Fixed Sof: C217(0.4) H217(0.4) C218(0.4) C219(0.4) H219(0.4) C220(0.4)
 H220(0.4) C221(0.4) H221(0.4) C222(0.4) H222(0.4) C223(0.4) H223(0.4)
 H22A(0.4) H22B(0.4) H22C(0.6) H22D(0.6) O72(0.5) O73(0.5) O74(0.5) Cl60(0.7)
 O61(0.7) O62(0.7) O63(0.7) O64(0.7) O52(0.5) O53(0.5) O54(0.5) N900(0.5)
 C901(0.5) O52A(0.5) O73A(0.5) O53A(0.5) O74A(0.5) O54A(0.5) C902(0.5)
 H90A(0.5) H90B(0.5) H90C(0.5) O72A(0.5) C1A(0.6) H1A(0.6) C1B(0.6) C1C(0.6)
 H1C(0.6) C1D(0.6) H1D(0.6) C1E(0.6) H1E(0.6) C1F(0.6) H1F(0.6) C1G(0.6)
 H1G(0.6) Cl6A(0.3) O61A(0.3) O62A(0.3) O63A(0.3) O64A(0.3)
6.a Ternary CH refined with riding coordinates:
 C7(H7), C30(H30), C49(H49), C72(H72), C91(H91), C114(H114), C133(H133),
 C156(H156), C175(H175), C198(H198), C217(H217), C240(H240), C1A(H1A)
6.b Secondary CH2 refined with riding coordinates:
 C14(H14E,H14F), C15(H15E,H15F), C28(H28A,H28B), C29(H29A,H29B), C56(H56A,
 H56B), C57(H57A,H57B), C70(H70A,H70B), C71(H71A,H71B), C98(H98A,H98B),
 C99(H99A,H99B), C112(H11A,H11B), C113(H11C,H11D), C140(H14A,H14B), C141(H14C,
 H14D), C154(H15A,H15B), C155(H15C,H15D), C182(H18A,H18B), C183(H18C,H18D),
 C196(H19A,H19B), C197(H19C,H19D), C224(H22A,H22B), C224(H22C,H22D), C225(H22E,
 H22F), C238(H23A,H23B), C239(H23C,H23D)
6.c Me refined with riding coordinates:
 C902(H90A,H90B,H90C)
6.d Aromatic/amide H refined with riding coordinates:
 C1(H1), C2(H2), C3(H3), C4(H4), C6(H6), C9(H9), C10(H10), C11(H11), C12(H12),
 C13(H13), C17(H17), C20(H20), C22(H22), C23(H23), C26(H26), C27(H27), C32(H32),
  C33(H33), C34(H34), C35(H35), C36(H36), C37(H37), C39(H39), C40(H40),
 C41(H41), C42(H42), C43(H43), C44(H44), C45(H45), C46(H46), C48(H48), C51(H51),
  C52(H52), C53(H53), C54(H54), C55(H55), C59(H59), C62(H62), C64(H64),
 C65(H65), C68(H68), C69(H69), C78(H78), C77(H77), C76(H76), C75(H75), C74(H74),
  C79(H79), C81(H81), C82(H82), C83(H83), C84(H84), C85(H85), C86(H86),
 C87(H87), C88(H88), C90(H90), C93(H93), C94(H94), C95(H95), C96(H96), C97(H97),
  C101(H101), C104(H104), C106(H106), C107(H107), C110(H110), C111(H111),
 C116(H116), C117(H117), C118(H118), C119(H119), C120(H120), C121(H121),
 C123(H123), C124(H124), C125(H125), C126(H126), C127(H127), C128(H128),
 C129(H129), C130(H130), C132(H132), C135(H135), C136(H136), C137(H137),
 C138(H138), C139(H139), C143(H143), C146(H146), C148(H148), C149(H149),
 C152(H152), C153(H153), C158(H158), C159(H159), C160(H160), C161(H161),
 C162(H162), C163(H163), C165(H165), C166(H166), C167(H167), C168(H168),
 C169(H169), C170(H170), C171(H171), C172(H172), C174(H174), C177(H177),
 C178(H178), C179(H179), C180(H180), C181(H181), C185(H185), C188(H188),
 C190(H190), C191(H191), C194(H194), C195(H195), C200(H200), C201(H201),
 C202(H202), C203(H203), C204(H204), C205(H205), C207(H207), C208(H208),
 C209(H209), C210(H210), C211(H211), C212(H212), C213(H213), C214(H214),
 C216(H216), C219(H219), C220(H220), C221(H221), C222(H222), C223(H223),
 C227(H227), C230(H230), C232(H232), C233(H233), C236(H236), C237(H237),
 C242(H242), C243(H243), C244(H244), C245(H245), C246(H246), C247(H247),
 C249(H249), C250(H250), C251(H251), C252(H252), C1C(H1C), C1D(H1D), C1E(H1E),
 C1F(H1F), C1G(H1G)
6.e Fitted hexagon refined as free rotating group:
 C73(C78,C77,C76,C75,C74)
6.f Idealised Me refined as rotating group:
 C702(H70C,H70D,H70E), C802(H80A,H80B,H80C)
;
_shelx_res_checksum              32053
_shelx_fab_checksum              88957
_olex2_submission_special_instructions 'No special instructions were received'
_oxdiff_exptl_absorpt_empirical_details
'Empirical correction (ABSPACK) includes: - Absorption correction using spherical harmonics - Frame scaling'
_oxdiff_exptl_absorpt_empirical_full_max 1.335
_oxdiff_exptl_absorpt_empirical_full_min 0.835