Page created by Vikram K. Mulligan (vmullig@uw.edu) on 27 November 2016.
Last updated on 18 October 2018.
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Description

This mover places chemical cross-linkers such as 1,3,5-tris(bromomethyl)benzene (TBMB) or trimesic acid (TMA). It can set up covalent bonds and constraints, pack and energy-minimize the linker and the side-chains to which it is connected, and relax the entire structure. Options are provided for filtering based on input geometry, to throw out poses that do not present side-chains in conformations compatible with the linker.

Metals are also effectively cross-links that connect several side-chains, as far as Rosetta is concerned, so as of 16 May 2018, this mover can also place metals. When placing metals, metal-coordinating residues (currently L- or D-histidine, glutamate, or aspartate) are patched to add a virtual atom on the metal-coordinating side-chain atom representing the metal, atom pair constraints are added to tether the virtual atoms to one another and to ensure that all of the virtual atoms overlap (representing a single metal ion), and angle constraints are added for each trio of (liganding atom 1)--(metal virtual atom)--(liganding atom 2) to enforce tetrahedral, octahedral, square pyramidal, square planar, trigonal pyramidal, or trigonal planar geometry. Future development will add support for other metal coordination geometries.

Needed flags

The CrosslinkerMover requires that Rosetta load a params file for the crosslinker, as well as the sidechain conjugation variant types for the sidechains that will be cross-linked. These are not loaded by default. For example, to link three cysteine residues with TBMB, one needs the following commandline flag:

-extra_res_fa sidechain_conjugation/CYX.params crosslinker/1.3.5_trisbromomethylbenzene.params

These load the CYX cysteine variant (used when cysteine is conjugated to things other than disulfide-forming residues) and the TBMB crosslinker.

When using the mover with symmetry, the symmetric variant of the TBMB crosslinker (which is one third of the crosslinker -- vide infra) must be loaded instead:

-extra_res_fa sidechain_conjugation/CYX.params crosslinker/1.3.5_trisbromomethylbenzene_symm.params

When placing metals, no special flags are needed.

Examples

Placing 1,3,5-tris(bromomethyl)benzene (TBMB)

This example places 1,3,5-tris(bromomethyl)benzene (TBMB), linking cysteine residues 7, 21, and 35 with the linker. It also sets up constraints, and repacks and energy-minimizes the linker and the side-chains to which it is connected. By default, no global relaxation is performed, however. In this case, the pose is asymmetric:

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
                <Index name="select_cys" resnums="7,21,35" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <CrosslinkerMover name="place_tbmb" residue_selector="select_cys" linker_name="TBMB" scorefxn="r15" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="place_tbmb" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

Placing a metal with trigonal planar or trigonal pyramidal coordination geometry

In this example, we assume that positions 12, 16, and 23 in the input structure are residue types that can coordinate a metal (currently, L- or D-histidine, aspartate, or glutamate). The metal_type option determines the liganding atom-metal bond lengths, and defaults to "Zn" (zinc). In the case of trigonal planar coordination, bond angles are constrained to 120 degrees, and an improper torsional constraint additionally pulls the metal into the plane of the liganding three atoms. In the case of trigonal pyramidal coordination, bond angles are constrained to 109.47 degrees, and no improper torsional constraint is applied.

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
                <Index name="select_metal_coordinating" resnums="12,16,23" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <!-- Note: in the definition below, "trigonal_planar_metal" may be replaced with "trigonal_pyramidal_metal". -->
                <CrosslinkerMover name="metal_xlink" residue_selector="select_metal_coordinating" linker_name="trigonal_planar_metal" metal_type="Zn" scorefxn="r15" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="metal_xlink" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

Placing a metal with square planar or square pyramidal coordination geometry

In this example, we assume that positions 3, 12, 16, and 23 in the input structure are residue types that can coordinate a metal (currently, L- or D-histidine, aspartate, or glutamate). The metal_type option determines the liganding atom-metal bond lengths, and defaults to "Ni2" (nickel in the 2+ oxidation state) in the case of square planar or square pyramidal geometries. In the case of square planar coordination, bond angles are constrained to 90 or 180 degrees, and an improper torsional constraint additionally pulls the metal into the plane of the liganding four atoms. In the case of square pyramidal coordination, bond angles are constrained to 90 or 180 degrees, an ambiguous improper torsional constraint is applied keeping the metal in the plane of any four of the liganding atoms.

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
		<!--
			A fifth position must be selected by the residue selector below in the case of square pyramidal
			metal coordination.
		-->
                <Index name="select_metal_coordinating" resnums="3,12,16,23" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <!--
			Note: in the definition below, "square_planar_metal" may be replaced with "square_pyramidal_metal".  In this case, a fifth residue
			must be selected by the select_metal_coordinating residue selector.
		 -->
                <CrosslinkerMover name="metal_xlink" residue_selector="select_metal_coordinating" linker_name="square_planar_metal" metal_type="Ni2" scorefxn="r15" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="metal_xlink" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

Placing a tetrahedrally-coordinated metal

In this example, we assume that positions 7, 12, 16, and 23 in the input structure are residue types that can coordinate a metal (currently, L- or D-histidine, aspartate, or glutamate). The metal_type option determines the liganding atom-metal bond lengths, and defaults to "Zn" (zinc). The CrosslinkerMover adds 109.47 degree bond angle constraints.

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
                <Index name="select_metal_coordinating" resnums="7,12,16,23" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <CrosslinkerMover name="metal_xlink" residue_selector="select_metal_coordinating" linker_name="tetrahedral_metal" metal_type="Zn" scorefxn="r15" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="metal_xlink" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

Placing an octahedrally-coordinated metal

In this example, we assume that positions 7, 12, 16, 23, 26, and 30 in the input structure are residue types that can coordinate a metal (currently, L- or D-histidine, aspartate, or glutamate). The metal_type option determines the liganding atom-metal bond lengths. Currently, for octahedral coordination, the only supported type is Fe2 (for iron(II)), though this will be expanded in the future. The CrosslinkerMover adds 90 or 180 degree bond angle constraints.

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
                <Index name="select_metal_coordinating" resnums="7,12,16,23,26,30" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <CrosslinkerMover name="metal_xlink" residue_selector="select_metal_coordinating" linker_name="octahedral_metal" metal_type="Fe2" scorefxn="r15" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="metal_xlink" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

Usage with symmetry

This mover can be used for the case of linkers with symmetry that matches the symmetry of the pose. In this case, the linker residue that gets added to the pose is actually a fragment of the total linker (for example, one third of the geometry in the case of C3 symmetry). N such residues are added, one to each symmetry repeat, and covalent bonds and suitable constraints are set up between the fragments. Such usage requires that the user pass the symmetry=<string> option, where the string is an uppercase character representing the symmetry type (C=cyclic, S=mirror cyclic, D=dihedral) followed by an integer indicating the number of repeats (e.g. "C3" for cyclic, threefold symmetry). Here is an example in which the CrosslinkerMover is used with a symmetric pose with C3 symmetry (defined in a symmetry definition file inputs/c3.symm). In this case, the ResidueSelector must select equivalent cysteine residues in the three symmetric copies.

<ROSETTASCRIPTS>
        <SCOREFXNS>
                <ScoreFunction name="r15" weights="ref2015.wts" symmetric="true" />
        </SCOREFXNS>
        <RESIDUE_SELECTORS>
                <Index name="select_cys" resnums="7,21,35" />
        </RESIDUE_SELECTORS>
        <MOVERS>
                <SetupForSymmetry name="setup_symm" definition="inputs/c3.symm" />
                <CrosslinkerMover name="threefold" residue_selector="select_cys" linker_name="TBMB" scorefxn="r15" symmetry="C3" />
        </MOVERS>
        <PROTOCOLS>
                <Add mover="setup_symm" />
                <Add mover="threefold" />
        </PROTOCOLS>
</ROSETTASCRIPTS>

When placing tetrahedrally-coordinated metals, compatible symmetries are C2, D2, and S4. When placing octahedrally-coordinated metals, compatible symmetries are C3, C2, D3, S2, and S6.

Full options

<CrosslinkerMover name="(&string)" scorefxn="(&string)" residue_selector="(&string)" linker_name="(&string)"
    add_linker="(true &bool)" constrain_linker="(true &bool)"
    pack_and_minimize_linker_and_sidechains="(true &bool)" sidechain_fastrelax_rounds="(3 &int)"
    do_final_fastrelax="(false &bool)" final_fastrelax_rounds="(3 &int)" symmetry="(A1 &string)"
    filter_by_sidechain_distance="(true &bool)" sidechain_distance_filter_multiplier="(1.0 &real)"
    filter_by_constraints_energy="(true &bool)" constraints_energy_filter_multiplier="(1.0 &real)"
    filter_by_final_energy="(false &bool)" final_energy_cutoff="(0.0 &real)" metal_type="(Zn &string)"
/>
Option Required Type Description
name YES string A unique name for this instance of the CrosslinkerMover.
linker_name YES string The name of the type of linker to use. Currently, the allowed options are "tetrahedral_metal", "octahedral_metal", "trigonal_planar_metal", "square_planar_metal", "trigonal_pyramidal_metal", "square_pyramidal_metal", "TBMB" (for 1,3,5-tris(bromomethyl)benzene), and "TMA" (for trimesic acid).
residue_selector YES string A previously-defined residue selector that has been set up to select the correct number of residues for the crosslinker type.
scorefxn YES string A scorefunction to use for packing, energy-minimization, and filtering. If constraints are turned off in this score function, they will be turned on automatically at apply time.
add_linker No bool Should the linker geometry be added to the pose? Default true.
constrain_linker No bool Should constraints for the linker be added to the pose? Default true.
pack_and_minimize_linker_and_sidechains No bool Should the linker and the connecting sidechains be repacked, and should the jump to the linker, and the linker and connnecting side-chain degrees of torsional freedom, be energy-minimized? Default true.
sidechain_fastrelax_rounds No int The number of rounds of FastRelax to apply when packing and minimizing side-chains and the liker. Default 3.
do_final_fastrelax No bool Should the whole pose be subjected to a FastRelax? Default false.
final_fastrelax_rounds No int The number of rounds of FastRelax to apply when relaxing the whole pose. Default 3.
symmetry No string Is this a symmetric pose being connected by a crosslinker with matching symmetry? Default unused if not specified. If specified, the string must be a single uppercase character representing the symmetry type ("C" for cyclic, "S" for mirror cyclic, "D" for dihedral, or "A" for achiral) followed by an integer representing the number of symmetry repeats (e.g. "S4" for cyclic fourfold symmetry with mirroring about the plane perpendicular to the symmetry axis from one repeat to the next).
filter_by_sidechain_distance No bool Prior to adding the linker geometry, should this mover abort with failure status if the selected side-chains are too far apart to connect to the linker? Default true.
sidechain_distance_filter_multiplier No real This is a multiplier for the sidechain distance cutoff filter. Higher values make the filter less stringent. Default 1.0.
filter_by_constraints_energy No bool After adding the linker geometry, adding constraints, and repacking and minimizing the linker and the connecting side-chains, should ths mover abort with failure status if the constraints energy is too high (i.e. the energy-minimized linker geometry is bad)? Default true.
constraints_energy_filter_multiplier No real This is a multiplier for the constraints energy cutoff filter. Higher values make the filter less stringent. Default 1.0.
filter_by_final_energy No bool At the end of this protocol, should this mover exit with error status if the final energy is above a user-defined cutoff? Default false.
final_energy_cutoff No real If we are exiting with error status if the final energy is too high, this is the energy cutoff. Default 0.0.
metal_type No string The type of metal. This determines the distance between the metal liganding atom and the virtual atom representing the metal. Defaults to "Zn" (for zinc). Only used when placing metal crosslinks.

Residue types that can be linked

Note that each type of crosslinker can link different types of side-chains:

Abbreviation Crosslinker Types that can be linked
octahedral_metal virtual atoms representing a metal (note: no new residue is placed) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
tetrahedral_metal virtual atoms representing a metal (note: no new residue is placed) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
square_pyramidal_metal virtual atoms representing a metal (note: no new residue is placed ) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
square_planar_metal virtual atoms representing a metal (note: no new residue is placed ) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
trigonal_pyramidal_metal virtual atoms representing a metal (note: no new residue is placed ) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
trigonal_planar_metal virtual atoms representing a metal (note: no new residue is placed ) L-histidine (HIS, HIS_D), D-histidine (DHI), L-aspartate (ASP), D-asparate (DAS), L-glutamate (GLU), D-glutamate (DGU)
TBMB 1,3,5-tris(bromomethyl)benzene L-cysteine (CYS), D-cysteine (DCY)
TMA trimesic acid L-lysine (LYS), D-lysine (DLY), L-ornithine (ORN), D-ornithine (DOR), L-2,4-diaminobutyric acid (DAB), D-2,4-diaminobutyric acid (DDA), L-2,3-diaminopropanoic acid (DPP), D-2,3-diaminopropanoic acid (DDP)

See also