Computational protocol: Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

Similar protocols

Protocol publication

[…] Human β-cardiac MD::GFP protein (1.5–2.0 mg ml−1) in 25 mM HEPES pH 7.0, 5 mM TCEP (Sigma) was screened against four Hampton Research (Aliso Viejo, CA) high-throughput crystallization screens using 0.2 μl protein and 0.2 μl mother liquor (ML) per drop equilibrated against 60 μl of ML at room temperature. Five drops containing brightly fluorescent birefringent crystals were found. These initial conditions were refined and single crystals without drug (Apo) were grown from 10% Tacsimate, pH 6.0, 14–15% PEG 3350, 10% glycerol, 0.2 mM MgCl2 and 5 mM TCEP. Crystals with OM (OM+) were grown in essentially the same condition with 125 μM OM added to the protein drop (0.5% DMSO). The crystallization conditions proved to be a suitable cryoprotectant; therefore, crystals taken directly from the ML were fast-cooled in liquid nitrogen for X-ray analysis.Single-crystal diffraction data for the Apo structure were collected on a Rigaku rotating anode X-ray generator (λ=1.542 Å) with a Raxis IV++ detector at 100 °K. The OM+ crystal diffraction data were collected at Brookhaven National Laboratory NSLS beamline X29A (λ=1.075 Å) with an ADSC QUANTUM 315 detector at 100 °K. Reflections were indexed, integrated and scaled with iMosflm and Aimless from the ccp4 package. The Apo structure was determined first by molecular replacement with Phaser using residues 4–780 of chicken skeletal muscle myosin subfragment 1 structure, PDB ID: 2MYS, together with the structure of GFP, PDB ID: 2QLE, as the search ensemble. The molecular replacement map clearly identified two motor domains associated with one GFP per domain. The helix linking the MD to the GFP was not included in the search models; however, new density corresponding to the helix was apparent in the initial maps. After building the linking helix into the density and joining the MD and GFP domains, the cMD and GFP sequence were built into the model with PHENIX Autobuild. The structure was refined with PHENIX refine and iterative model building in Coot. Only a very limited number of well-defined water molecules and two sulfate anions in the cMD β-phosphate-binding loop were included in the structure at this resolution. The statistics for favoured, allowed and outlier Ramachandran angles are 93.4%, 6.1% and 0.5%, respectively. The Molprobity score for the refined structure is 2.16 and the coordinates are deposited as PDB ID: 4P7H.The OM+ structure was solved by molecular replacement using the motor domain (2–708) of the Apo structure A chain and the helix–GFP domain as separate models in the search ensemble followed by PHENIX Autobuild. After limited initial refinement, unassigned density likely coming from the ligand was apparent. PHENIX LigandFit was used to search and dock OM into the map using Resolve. Ligand restraints for refinement were generated with eLBoW. The structure was refined as with the Apo structure. The statistics for favoured, allowed and outlier Ramachandran angles are 95.3%, 4.2% and 0.5%, respectively. The Molprobity score for the refined structure is 1.87.The Karplus and Diederichs CC* data and model quality-assessment tool were applied to both models and data sets to assess the high-resolution cutoff. The Apo data set was truncated at 3.2 Å, with mean (I/σ(I))=2 and a CC*>0.88 for the highest-resolution shell (3.3–3.2 Å). The CC* statistic for the OM+ data is >0.73 for the highest-resolution shell (2.33–2.25 Å), supporting the inclusion of the full data range in determination of the model. Alternatively, applying traditional cutoff criteria of the mean (I/σ(I))≥2 to the OM+ data would make this a nominal 2.40-Å resolution structure. The coordinates for the OM+ structure are deposited as PDB entry 4PA0 (ref. ). Data collection and refinement statistics are summarized in . […]

Pipeline specifications

Software tools iMosflm, PHENIX, Coot, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Heart Diseases, Heart Failure
Chemicals Nucleotides, Phosphates