Comparative Protein Structure Modeling Using Modeller

Abstract

Supplemental methods Homology ModellingThe homology model of the NatA WT complex was built with Modeller version 9.8 (1) using the NatA complex of S. pombe as a template. The sequence identity between the target and the template is around 67% and 37% for the subunits Naa10 and Naa15, respectively (Figure S1). The sequence alignments were obtained using ClustalW. Thirty models were generated and evaluated using the Discrete Optimized Protein Energy potential (DOPE score). The models with the lowest overall DOPE score were selected. The S37P NatA complex was designed from the human WT NatA complex using SCWRL instead of Modeller, in order to have starting structures as similar as possible as to avoid bias in the comparison between the wild type and mutant NatA complex. System PreparationPROPKA (2) was used to determine the protonation state of histidines. All other titratable groups were modelled in their standard protonation states. Hydrogen atoms were constructed using the HBUILD module of the CHARMM program (3). The complexes were solvated in cubic boxes of TIP3P water (4) with 120 Å-long edges. Water molecules overlapping the proteins, determined by a cut-off of 2.8Å, were removed. Molecular dynamicsMolecular dynamics (MD) simulations were used to explore the conformational space. As the aim was to uncover differences between the two systems, we ran long simulations (100 ns) in order to allow the systems to rearrange. These simulations were performed at a temperature of 300K using the NAMD program (5) and the CHARMM27 force field (4). The SHAKE algorithm was used to constrain all bonds between hydrogen and heavy atoms. Non-bonded interactions were truncated at a cut-off of 14Å, using a switch function for both the van der Waals, and electrostatic interactions (6). The particle-mesh Ewald algorithm (7) was used to evaluate the long range electrostatic interactions. The system was subjected to an energy minimization of 1000 steps using the conjugated gradient algorithm, followed by a gradual heating consisting of four successive simulations at temperatures of 10K, 100K, 200K and 300K. This was followed by a 1 ns equilibration phase during which velocities were reassigned every picosecond. The production phase consisted of a 100 ns simulation in the NPT ensemble, with a time step of 1fs. Two simulations (replicas) using a different set of

Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase—A QM/MM Study

“…Ala1-Asp2-Leu3 and Asp357, were added with the program Modeller. [29][30][31][32] The ferrous ion was substituted with an iron-oxo group and acetate was replaced by HPA. Protonation states (at pH 7.0) of basic and acidic residues (other than iron ligands), were assigned based on pK a calculations performed with the PROPKA server, 33,34 with no unexpected protonation states predicted.…”

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

“…Homology Model-The human GGT homology model was constructed using MODELLER version 9 software (38). The large subunit of GGT was generated using Escherichia coli GGT (Protein Data Bank code 2DBW), which exhibits 29% sequence identity, as a template (39).…”

The cohesin ring concatenates sister DNA molecules

“…S2). We created a homology model 11 of the yeast Smc1/Smc3 hinge heterodimer based on the homodimeric Thermotoga maritima crystal structure 4 and identified two juxtaposed side-chains that we mutated to cysteines. Incubation of the engineered Smc1/Smc3 hinge dimer with either bBBr or BMOE caused efficient cross-linking within a few minutes (Fig.…”