Computational protocol: Importance of the C-terminal histidine residues of Helicobacter pylori GroES for Toll-like receptor 4 binding and interleukin-8 cytokine production

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Protocol publication

[…] SAXS measurements were collected at beamline 23A1 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan, with an online size-exclusion high pressure liquid chromatographic (SE-HPLC) system. Typically, 50 μL protein sample (20 mg mL−1) was injected into the HPLC column. Next, the sample was directed into a quartz capillary for SAXS measurements thermostatted at 298 K. Data were collected at a rate of 1 frame per 30 s using a Pilatus 1M-F area detector. The energy of the X-ray beam was 15 keV (wavelength λ = 0.8266 Å), and the setup adjusted to achieve scattering q values of 0.007 Å−1 to 0.3 Å−1, q defined by 4πλ−1sinθ with scattering angle 2θ. Data were corrected for electronic noise, and sample transmission, followed by scaling to absolute intensity I(q) in units of cm−1 via scattering from water at protein sample conditions. Scattering curves for each protein were used for Guinier analyses using the program Primusqt from the ATSAS package. The pair distribution functions P(r) calculated the radius of gyration (Rg) by integrating the function with r2 over all values of r and maximum particle dimensions (Dmax) by computing with autoGNOM. Rg estimates were also obtained using Guinier analysis. Ab initio modeling was performed with the program DAMMIN to obtain 10 to 15 independent dummy bead models that were subsequently averaged in DAMAVER software. Bead models of molecular envelopes for each structure were converted to surfaces with Chimera. Each DAMMIN model showed good fit to the experimental SAXS curve (χ2 = 1.4~2.1) and was calculated with normalized spatial discrepancy (NSD) values in the range 0.5–0.7. To construct the conformation of domain B missing from other crystal or solution structures, CORAL models of WT and histidine mutants were also built based on the domain A template of HpGroES from SWISSMODEL with Mycobacterium tuberculosis GroES (PDB: 1P3H) assembled with 28 dummy C-terminal residues. Rigid body modeling into SAXS-derived molecular envelopes was manually performed using the program Chimera. […]

Pipeline specifications

Software tools ATSAS, DAMMIN
Application Small-angle scattering
Organisms Helicobacter pylori, Escherichia coli
Diseases Stomach Neoplasms, Stomach Ulcer, Helicobacter Infections
Chemicals Nickel