JLigand tutorial (new monomer)

This tutorial uses a 1.15 Å resolution P2₁2₁2₁ crystal structure RNase Sa. The asymmetric unit contains two protein molecules of which only one binds an inhibitor 2'GMP in its active site.

We assume that we are at a late stage of model building. The current model of the crystal structure includes only two protein molecules and we need to generate the description and coordinates of 2'GMP, add this ligand to the incomplete model and refine the new model. The whole procedure includes the following four steps.

• Making description of 2'-GMP (JLigand)

• Calculating phases (REFMAC)

• Fitting ligand into the density (Coot)

• Refinement of structure with ligand (REFMAC)

Download required data from here. Uncompress the downloaded file to obtain the directory JLigand_comp_tutorial containing files data.mtz and model.pdb.

The file data.mtz contains columns FP and SIGFP taken from $CCP4/examples/tutorial/data/rnase115.mtz and, in addition, the FREE column.

The file model.pdb was generated from $CCP4/examples/tutorial/data/rnase-3gp.pdb. All non-protein residues were removed and this incomplete model was refined against data.mtz.

Making description of 2'-GMP

Actions (indented text) are performed in JLigand window except when the window is explicitly specified (underlined text).

• Start JLigand in the directory JLigand_comp_tutorial:

Terminal: cd JLigand_comp_tutorial

Terminal: jligand

• Load 3'-GMP; its three-letter code is 3GP:

Load Ligand: type in 3GP, press enter and wait a bit

• Open a help window, the one that is appropriate to your computer setup:

Help > JLigand Mouse Help or

Help > JLigand Keypad Help

• Play with the molecule: rotate (hold the left mouse button and drag), move (hold right mouse button and drag) and zoom (rotate mouse wheel).

• Show atom names:

Tick Atom Ids check box (in the second horizontal panel from the top)

Note: Only the first letter of atom label is clickable; it is underlined when cursor is moved to it.

• Play with an atom: edit atom data (double-click on the atom, i.e. on the first letter of atom label), delete (press Del in the left panel and click on atom) and undo (press Undo button in the second line of the top panel).

• Delete the bond between O3* and P:

Press Del in the left panel and click on the bond

• Add a single bond between O2* and P:

Select Link in the left panel; click on one atom and then on the other

• Change the ligand's Id from 3GP to e.g. XMP:

Double click on the label 3GP, which is below the picture of ligand

Edit Ligand Id: type in XMP and Submit

• Regularise the ligand:

Ligand > Regularise > XMP

• Show hydrogen atoms (you may want to zoom in the picture to have a better view):

Tick Hydrogens check box

• Check that hydrogen atoms were properly handled during regularisation, i.e. that HO2* was removed and HO3* was added. Note that sometimes hydrogen atoms need to be removed explicitly before the regularisation, e.g. when a single bond is being replaced with a double bond.

• Save a description of XMP:

File > Save as Monomer > XMP

Save as CIF-library: Save

Info: OK

• Have a look at the description of the file that has been created:

View > View File...

View File: select XMP.cif and press View

• Save coordinates of XMP excluding hydrogen atoms (REFMAC will automatically add hydrogen atoms according to the description of XMP if requested, but a ligand without hydrogen atoms is easier to handle in Coot):

File > Save Coordinates > XMP

Save Coordinates: select Exclude Hydrogens and press Submit

Save Coordinates in PDB-file: keep the default File: XMP.pdb and press Save Coordinates

Info: OK

• All required files have been created and JLigand can now be quit:

JLigand > Quit

Quit JLigand: Yes

Calculating phases

At this point we need to generate an mtz-file with phases. This file will be used to draw the electron density maps in Coot.

• Run REFMAC with zero cycles of refinement, and with input files model.pdb and data.mtz. This can be done using CCP4I or you can copy and paste the following four lines into the command line:

refmac5 xyzin model.pdb hklin data.mtz xyzout refmac1.pdb hklout refmac1.mtz

make hydr no

ncyc 0

end

Fitting ligand into the density

• Start Coot and open files refmac1.pdb and refmac1.mtz:

Terminal: coot

Coot: File > Open Coordinates...

Select Coordinates File: select refmac1.pdb; OK

Coot: File > Auto Open Mtz...

Select Dataset File: select refmac1.mtz; OK

• Load the description of XMP from CIF-library file and coordinates from PDB-file:

Coot: File > Import CIF dictionary

Select File: select XMP.cif and press OK

Coot: File > Open Coordinates

Select Coordinates File: select XMP.pdb and press OK

• Fit XMP into its density:

Coot: Calculate > Other Modelling Tools

Other Modelling Tools: select Find Ligands and Close

Find Ligands: Enlarge this window; in Select Ligands to Search for: tick .../XMP.pdb and Flexible? and then press Find'em! button

New Ligands: OK

Fitted Ligands: press Fitted Ligand #0 and then OK

• Open Model/Fit/Refine window:

Coot: Calculate > Model/Fit/Refine

All the tools presented in this window are also presented in the right panel of Coot window; we use the Model/Fit/Refine window because it contains both icons and text references

• Correct the torsion angle C2*-C1*-N9-C8. Skip this step if the adenine base has correct orientation, otherwise rotate the base about the bond C1*-N9:

Coot: Find required atoms (double-click on atom to display its label)

Model/Fit/Refine: select Torsion General

Coot: click on atoms C2*, C1*, N9, C8 in this order

Coot: keep left mouse button pressed and drag the mouse to rotate the base

Accept Refinement?: Accept

• Refine fitting of XMP:

Model/Fit/Refine: Select Map

Select Map for Fitting: OK

Model/Fit/Refine: Real Space Refine Zone

Coot: Click twice on the ligand.

Coot: Keep left mouse button pressed and drag atoms into their density.

Coot: To handle an individual atom, move cursor to it, keep ctrl-key and left mouse button pressed and drag the atom into its density.

Accept refinement: Accept

Model/Fit/Refine: Close

• Append fitted ligand to the protein molecule:

Coot: Calculate > Merge Molecules...

Merge Molecules: Enlarge this window; in Append/Insert Molecule(s): tick Fitted ligand # 0, and then press Merge

• Should you want to continue with model correction, it would make sense to remove used objects:

Coot: Display Manager

Display Manager: Enlarge this window to see all the molecules and Delete Molecule buttons.

Display Manager: Unselect Display check box for Fitted ligand # 0. If the copy of fitted ligand is there in the graphics window then previous steps have been done correctly and you can proceed and delete used objects.

Display Manager: In section Molecules: press Delete Molecule next to XMP.pdb and Fitted ligand #0. In section Maps: press Delete Molecule next to Masked (by protein).

Display Manager: Close

• Save the merged molecule:

Coot: File > Save Coordinates...

Save Coordinates Molecule Selector: keep default selection .../refmac1.pdb and press Select Filename

Select Filename: keep default selection refmac1-coot-0.pdb and press OK

• The molecule with appended ligand is saved into the file refmac1-coot-0.pdb and Coot can be exited:

Coot: File > Exit

Refinement of structure with ligand

• Run REFMAC with the new coordinate file and previously created additional library XMP.cif. Use CCP4I, or copy and paste the following four lines into the command line:

refmac5 xyzin refmac1-coot-0.pdb hklin data.mtz libin XMP.cif xyzout refmac2.pdb hklout refmac2.mtz

make hydr no

ncyc 5

end

• Notice the decrease in R-free compared to the previous REFMAC run. (The values of R-factors can be found in the headers of PDB-files refmac1.pdb and refmac2.pdb.)

• Use Coot to examine the electron density of the ligand. (Start Coot; open refmac2.pdb and refmac2.mtz; navigate to the residue 1 of chain C using Draw > Go To Atom menu.)