Modeling Ligands into Maps Derived from Electron Cryomicroscopy

We are interested in understanding the physiological processes that occur across the cell, particularly at the membrane interface. We use cryoEM as a main technique and work on various interesting and challenging biological problems, studying multiple macromolecular complexes. Determining the structures of membrane proteins and other labile complexes was challenging couple of decades ago.

However, technical advances in cryoEM and novel detergent and lipid mimetic environments paved the way for rapid structure determination of these complexes. These structures can now be obtained at high resolution and potentially used in drug discovery. In single-particle cryoEM, challenges include acquiring stable and homogeneous sample, embedding the sample in random orientation in thin ice, processing a large number of images with low signal-to-noise ratio, and accurately determining the angular orientation and classification during 3D reconstruction.

Identifying and modeling small molecules in cryoEM maps of ligand protein complexes is challenging due to inherent noise in the data and isotropic and low map resolution, sample heterogeneity, and ligand flexibility. This protocol is a step-by-step guide for identifying and modeling ligands and solvent molecules in low-to-medium resolution cryoEM maps. Begin by downloading the unsharpened half maps of the apo-enolase from additional data in EMDB.

Then open ChimeraX and click on Open in the toolbar. Select the half maps of apo-enolase. Type this in the command line to combine the half maps and obtain the apo-enolase unsharpened map.

Adjust the threshold of the map to 0.0595 to reduce noise. Then type this command to rename the combined map. Click on Open.

Select the PEP enolase unsharpened half maps and type this command in the command line. Adjust the threshold of the map to 0.0721 to reduce noise and rename the map. Then display the apo-enolase and PEP enolase unsharpened maps and click on Fit in the map panel to compute a difference map.

Type this command in the command line to subtract the PEP enolase map from the apo-enolase map and adjust threshold. Color the subtracted map in green, denoting positive density. Next, open PDB and download the coordinates of Mycobacterium tuberculosis octomeric enolase.

Then in ChimeraX, click on Open from the toolbar and select the file name. Click Molecule Display, Hide Atoms, and Show Cartoon. Type this command to rename the model.

Select the model from the model panel. Click on Right Mouse from the toolbar. Then click Move Model and position the model in proximity to the PEP enolase map.

Align the model with respect to the map. Fit this model into the PEP enolase unsharpened map by typing this command in the command line. Check the fit.

Adjust the color and structure representation. Open Coot from the terminal by typing coot Click on File, then Open Coordinates, and select Apo-enolase.pdb. Next, download the PEP-bound enolase sharpened map from EMDB.

Display this map in Coot by clicking on File and Open Map. Scroll the middle mouse button to set the threshold to 7.00 sigma. Click on Validate, then Unmodeled Blobs.

In the new window, type 7.00 as the RMSD and click Find Blobs. Locate the unmodeled ligand density near the active site residues serine-42, lysine-386, and arginine-364. Next, click on File, Get Monomer, and enter PEP to get the PEP monomer model file from the Coot monomer library.

Move the PEP molecule to the density using the rotate/translate zone chain molecule option in the sidebar menu. Then click on real space refine zone in the sidebar menu to fit the PEP molecule in the density. Assess the fit and click on Accept when done.

Use the Merge Molecule option in the Edit tab to merge the fitted ligand with the apo-enolase model and save the model as pep-enolase.pdb. Then click on Calculate, NCS tools, followed by NCS Ligands to add ligands to the rest of the monomers in the coordinate file. For the protein with NCS option, select the coordinate file apo-enolase.

pdb and chain ID A as NCS master chain. Next, for the molecule containing ligand, select apo-enolase. pdb as the coordinate file, chain ID J and residue number 1 to 1.

Click on Find Candidate Positions. Click on the individual candidate ligands and analyze the fit visually. Then model two magnesium ions in the active site density by clicking on place atom at the pointer and selecting Mg from the pointer atom type list.

Also, add the magnesium ions in the symmetry-related monomers. Assess the fit for the symmetry-related magnesium atoms. Save the model by clicking on File, then Save Coordinates, Select Filename, and typing Enolase+PEP+Mg.pdb.

Then open Phenix GUI. Run a real-space refinement job using default parameters with the saved model and PEP-bound sharpened map. To visualize the modeled ligand, open PyMOL.

Click on File and Open to load the refined model from the Phenix refinement job. Also load the PEP-bound sharpened map. Rename the map as PEP-sharpened by clicking on Actions and then rename.

Then select the ligands by clicking on Display followed by Sequence. Type this in the command line and press enter. This carves the map density around the selection.

Click on the last box, C, next to the mesh_ligand object to change the color of the ligand mesh to blue. Display the active site residues interacting with the PEP and magnesium ions in stick-and-sphere representation and enolase enzyme in cartoon representation. Set mesh size.

Type this in the command line to perform ray tracing and save the image as a PNG file. During glycolysis, enolase converts 2-phosphoglycerate to phosphoenolpyruvate, a vital intermediate for various metabolic pathways. The difference map of the apoenzyme and PEP-bound enzyme showed a distinct ligand density.

Also, extra density was observed in the active site of the modeled protein. Phosphoenolpyruvate and two magnesium ions were modeled in the density observed in the vicinity of the ligand. Phosphoenolpyruvate formed hydrogen bonds with several active site residues, such as lysine-386 and arginine-364.

The magnesium ions formed metal coordination bonds with aspartate-241, glutamate-283, aspartate-310, and the phosphate of phosphoenolpyruvate.