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SubsystemInitializationNote: Neighborhood 2 (N2) is the most current version of the subsystem. For information on Neighborhood 1 (N1), see (here). Note 2: This page contains background about the development of Neighborhood 2. Feel free to skip ahead to Substitutions and Modifications to begin your experiments. While the best simulation would involve running MD on the entire ribosome, the PDB structures we are working with contain nearly 20,000 residues (more than 200,000 atoms). We simply do not have the computing power necessary to run the required calculations for each atom for a system of this size. Instead, we carve out a 494-residue subsystem that represents 2.6% of the total residues of the ribosome and 5.1% of the total atoms. There are a total of 38 chains in N2. N2 Design Step 1: Analogous to N1, a 40 angstrom sphere around the N3 atom of C1054 was selected in order to center the subsystem around the CAR, with a focus on the potential importance on stacking between of the anticodon and the CAR. However, this selection proved to be too large, capturing about 1000 residues. PyMOL syntax: select /5jup/A/A/C`1274/N3 around 40 Step 2: A 35 angstrom sphere around N3 of C1054 was selected, yielding a more appropriate ~600 residues. However, PyMOL only grabs the atoms in range (EXCLUDING N3 of C1054) leading to incomplete residues. Step 3: The whole-residue version of the 35 angstrom sphere was created. Additionally, residues were added in to minimize chain breaks and extend to natural chain ends if possible. Then the proto-onion shell was mapped by creating layers at different distances from C1054 using PyMOL's "extract" option. The layers used were 27-30A, 30-32A, and 32-35A. This left a 27A sphere of unrestrained residues around C1054. Step 4: The proto-onion shell was filled in with a few additional residues to decrease the number of transitions between restrained and unrestrained residues. Step 5: Areas where the proto-onion shell was thick were trimmed away, where removal of the 32-35A layer and shortening of artificial chain ends were preferred. We were also being careful not to open up artificial holes in the shell, adding a handful of residues to the onion shell if needed. Step 6: Finally, we considered modifications of ribosomal residues in the unrestrained portion of the system. We used a rRNA Database to map out 8 unrestrained residue nucleotide modifications. Research on additional modifications and past studies added the N4-acetylation of C1280 and the methylation of R146 to the list. Our final set of 10 subsystem modifications were: mR146, Um578, Y1187, m1acp3Y1191, Um1269, Gm1271, ac4C1280, Gm1428, Cm1639, and Am2256. N2 Preprocessing The PDB Processor was used to extract N2 from 5JUP. Next, the subsystem had 5' phosphates removed for compatibility with t-LEaP. This PDB was then edited by hand to substitute the +2 codon mRNA from:
All substitutions and modification experiments should begin with the unmodified N2 structure so that slightly different versions of t-LEaP (across Amber versions and running environments) don't cast doubt on our results. The path to this PDB is listed below. N2 Residue Table
PDB Processor: /home66/kscopino/SUBSYSTEM_DESIGN/PDB_Processor Remove 5' Phosphates: /home66/kscopino/SUBSYSTEM_DESIGN/rm_po4.py 5JUP_N2 Starting Structures (unmodified): GCU at +1 Codon: /home66/kscopino/SUBSYSTEM_DESIGN/5JUP_N2_GCU.pdb CGU at +1 Codon: /home66/kscopino/SUBSYSTEM_DESIGN/5JUP_N2_CGU.pdb Continue on to Substitutions and Modifications to begin your experiments! |