Computational protocol: A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

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Protocol publication

[…] Crystals were assessed for diffraction quality at the Stanford Synchrotron Radiation Lightsource (SSRL) beamlines 9-2, 11-1, and 12-2 and the Life Sciences Collaborative Access Team (LS-CAT) beamlines ID-21-D/F/G. Datasets were collected using the beamlines, temperatures, wavelengths, and detectors listed in and . All data were processed using the HKL2000 and CCP4 suites of programs. [...] A Dy3+ derivative was prepared by soaking pre-formed crystals of GspBBR in 1 mM DyCl3 for three days. Data were collected at three wavelengths near the Dy3+ L3 edge (). Dy3+ bound to a single site in the protein as determined using the SHELXD subroutine in the program SHARP . While HoCl3 also successfully derivatized GspBBR, non-isomorphism between crystals prevented the use of this second derivative in a traditional MIR calculation. The non-isomorphism was so severe that phases calculated in SHARP only used data from a single DyCl3 soaked crystal and did not include a native dataset for reference. This process resulted in reasonable phasing statistics and an overall figure of merit of 0.83 at 2.0 Å resolution (). Phases were improved by solvent flattening in DM which produced electron density maps of high quality (). Automated chain tracing was performed using PHENIX , which was able to trace residues 251–316 and 327–601, representing 94.7% of the model. This resulted in an initial Rcryst of 23.8% and Rfree of 25.5%.The lack of isomorphism between crystals prevented transfer of these initial coordinates to other data sets using a simple rigid body refinement. As a result, the model from the Dy3+ data set was transferred to the remaining data sets using the program PHASER followed by rigid body refinement in CNS . Each structure was subjected to alternate rounds of model building using the program COOT and refinement using CNS and REFMAC .The coordinates for the α-2,3-sialyl (1-thioethyl)galactose were built using CCP4i Sketcher and PRODRG . Refinement statistics for all final models are listed in . Figures were created using PyMOL , and inter-domain rotations were determined using DynDom . […]

Pipeline specifications

Software tools CCP4, SHELX, PHENIX, CNS, Coot, PRODRG, PyMOL, DynDom
Applications Drug design, Protein structure analysis
Organisms Streptococcus gordonii, Homo sapiens, Dipturus trachyderma
Diseases Endocarditis
Chemicals Carbohydrates