Computational protocol: A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

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Protocol publication

[…] All datasets were collected at 100 K on a Rigaku FR-E+ SuperBright rotating anode generator with VariMax optics and a Saturn 944 detector, at the Advanced Photon Source beamline 21-ID-F with a Marmosaic 225 CCD detector, or at the Canadian Macromolcular Crystallization Facility beamline 08ID-1 with a Marmosaic 225 CCD detector. All data were reduced with XDS/XSCALE []. The structure of MCL1 173–321 was phased by molecular replacement using Phaser from the CCP4 suite of programs with PDB ID 4OQ6 used as a search model [–]. Apo MBP-MCL1 was phased by molecular replacement using Phaser from the CCP4 suite of programs with MCL1 173–321 structure and 4MBP as search models. Subsequent structures of MBP-MCL1 were solved by utilizing the phases from the apo MBP-MCL1 structure and performing rigid body refinement in REFMAC []. All structures were completed using multiple cycles of refinement in Phenix followed by manual rebuilding of the structure using Coot [–]. All structures were quality checked by Molprobity []. Some of the data reported was supported by the SBGrid software platform []. All data reduction and refinement statistics are shown in and have been deposited in the PDB (PDB IDs: 4WMR, 4WMS, 4WMT, 4WMU, 4WMV, 4WMW, 4WMX). Fig.s, overlays, and electrostatic surface potentials were created using Pymol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.) […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Maltose