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In this step you will begin with the initial subsystem PDB file listed at the bottom of the page and make all necessary substitutions/modifications before bringing it through tLEaP. It is important to begin with this PDB at the beginning of each experimental construct rather than making successive modifications to reduce compounding errors and idiosyncrasies.

PDB Columns, referenced below

     1        2       3        4     5     6       7       8           9        10       11
     ATOM     265     HO5'     U     3     172.950 259.409 225.896     1.00     0.00     H

Nucleotide Substitutions

t-LEaP has the ability to grow in missing atoms when provided with a residue identity based on standard geometries within its library. In order to preserve the original orientation of the nucleotide when the substitution takes place, as many common atoms as possible should be conserved. Practically speaking, this requires going in and changing the subsystem .pdb. The file to use is linked at the bottom of the page.

The first step is to identify the residue(s) you will be changing. You can look up the residue number (PDB Column 5, above) using the following table:

Organism/Chain	Organism Identifier	Yeast Identifier	N1 Residue Number	N2 Residue Number
E. coli 16S	C1054	C1274	105	94
E. coli 16S	A1196	A1427	114	127
S. cerevisiae rps3	R146	R146	368	240
tRNA (A-site wobble, G)	nt 34	nt 34	167	408
tRNA (A-site nt2, G)	nt 35	nt 35	168	409
tRNA (A-site nt1, G)	nt 36	nt 36	169	410
mRNA (A-site nt1, C)	NA	NA	175	423
mRNA (A-site nt2, C)	NA	NA	176	424
mRNA (A-site wobble, U)	NA	NA	177	425
mRNA (+1 nt1, G)	NA	NA	178	426
mRNA (+1 nt2, C)	NA	NA	179	427
mRNA (+1 nt3, U)	NA	NA	180	428

For the nucleotide to be substituted/the residue number(s) you just identified, you will make the following changes:

Atoms of the sugar-phosphate backbone (P, OP1, OP2, and any atom with a terminal ' in PDB Column 3, above) should be retained. Non-common atoms of the base only (AKA atoms of the base that do not appear in the Common Atoms table below) should be deleted. For all remaining nucleotide atoms, the column that contains the residue identifier (PDB Column 4, above) for the residue to be substituted needs to be changed (to A, U, G, C, or INO). Lines that contain common atoms of the base should be updated with the correct atom identity (PDB Column 3, above) using the Common Atoms table below. If the atom changes element (i.e. from a C to an O), the last column (PDB Column 11, above) will also need to be modified.

Note that for inosine (INO in the table below) you should delete ANY hydrogen atoms in the residue being modified and the OP1 and OP2 atoms.

Common Atoms

C<->(A or INO)	C<->U	C<->G	U<->(A or INO)	(A or INO)<->G	U<->G
N1<->N9	N1	N1<->N9	N1<->N9	N1	N1<->N9
C6<->C8	C2	C6<->C8	C6<->C8	C2	C6<->C8
C2<->C4	O2	C2<->C4	C2<->C4	N3	C2<->C4
	N3			C4
	C4			C5
	N4<->O4			C6
	C5			N7
	C6			C8
				N9

Modifications

The first step is to identify the residue(s) you will be modifying. You can look up the residue number (PDB Column 5, above) using the following table:

Yeast Numbering	E. coli Numbering	N2 Residue Number	Modification Type
18S U578	16S U531	U28	2-O'-Me
18S U1269		U89	2-O'-Me
18S G1271	16S C1051	G91	2-O'-Me
18S G1428	16S G1197	G128	2-O'-Me
18S C1639	16S C1402	C173	2-O'-Me
25S A2256		A288	2-O'-Me
18S U1187	16S C962	U65	Ψ
18S U1191	16S G966	U69	m1acp3- Ψ
18S C1280		C100	ac4
Rps3 R146		R240	ω-NG-monomethyl

rRNA modifications have been added to the Amber standard library in the leaprc.modrna08 file (see tLEaP page). However, some of the atoms do not follow the same naming convention as the PDB files we have been using. First, you should delete ANY hydrogen atoms in the residue being modified. For the sugar-phosphate backbone, you should delete the OP1 and OP2 atoms.

For the base, you will need to bring the structure through tLEaP to make note of the naming convention. First, save a copy of the PDB you have at this stage. Then delete all the base atoms (anything NOT P, OP1, OP2, or any atom with a terminal '), change the column 4 residue identity to the correct three letter code (found in Table 3 of the 2007 Santalucia force field publication), and bring through tLEaP. Look at the modified residue of the resulting PDB for the modified base naming convention. Compare to the PDB copy you saved before you deleted any base atoms. Update the atom identities (column 3, last column) of the base from the copy to match the new PDB you generated, deleting any non-common atoms of the base if there are any. This will allow us to retain the original Cryo-EM residue geometry as much as possible.

Post-translationally modified amino acids may also need to be grown in and parameterized but are not a part of Amber's standard library. If the changed residue does not have a force field modification published by a separate group (check here, and here), it will have to be parameterized using antechamber.

Antechamber

Often drug design involves novel molecules that have never been computationally modeled. For residues that have not been parameterized, the antechamber module of Amber analyzes the molecule and creates a force field modification from the Generalized Amber Force Field (GAFF). If GAFF is unable to generate the force field modification, you will have to parameterize the residue yourself using quantum mechanical calculations (see the Amber manual). This then allows tLEaP to assign parameters to the residue.

Paths to Files

5JUP_N2 Starting Structure (unmodified):
   /home66/kscopino/SUBSYSTEM_DESIGN/5JUP_N2_GCU.pdb
      and /ribosome/Molecular Dynamics/Tutorials and Starting Structures/5JUP_N2_GCU.pdb
   /home66/kscopino/SUBSYSTEM_DESIGN/5JUP_N2_CGU.pdb
      and /ribosome/Molecular Dynamics/Tutorials and Starting Structures/5JUP_N2_CGU.pdb
Tutorials (on Google Drive):
   /ribosome/Molecular Dynamics/Tutorials and Starting Structures/Part1_tLEaP.mp4
   WITH CORRECTION:
   /ribosome/Molecular Dynamics/Tutorials and Starting Structures/Part1_tLEaP_correction.mp4