Modeling Ligands into Maps Derived from Electron Cryomicroscopy

Summary

This protocol introduces the tools available for modeling small-molecule ligands in cryoEM maps of macromolecules.

Abstract

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu₅, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied β-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.

Introduction

Cells accomplish their functions by carrying out innumerable chemical reactions simultaneously and independently, each meticulously regulated to ensure their survival and adaptability in response to environmental cues. This is achieved by molecular recognition, which enables biomolecules, especially proteins, to form transient or stable complexes with other macromolecules as well as small molecules or ligands¹. Thus, protein-ligand interactions are fundamental to all processes in biology, which include the regulation of protein expression and activity, the recognition of substrates and cofactors by enzymes, as well as how cells perceive and rel....

Protocol

1. Modeling phosphoenolpyruvate (PEP) in enolase from Mycobacterium tuberculosis

1. Identification of ligand density in the cryoEM map of PEP-enolase complex
   1. Download the unsharpened half-maps (emd_30988_additional_1.map and emd_30988_additional_2.map) of apo-enolase from the additional data in EMDB (see Table of Materials).
   2. Open ChimeraX (see Table of Materials). Open the half-maps of apo-enolase by clicking on Open

Discussion

The improvements in microscope hardware and software have resulted in an increase in the number of cryoEM structures in recent years. Although the highest resolution achieved at the moment in single particle cryoEM is 1.2 Å^57,58,59, the majority of the structures are being determined around 3-4 Å resolution. Modeling ligands in medium to low-resolution maps can be tricky and often fraught with ambiguity. Given the wide.......

Materials

Name	Company	Catalog Number	Comments
CCP4-8.0	Consortium of several institutes	https://www.ccp4.ac.uk	Free for academic users and includes Coot and list of tools developed for X-ray crystallography
CCP-EM	Consortium of several institutes	https://www.ccpem.ac.uk/download.php	Free for academic users and includes Coot, Relion and many others
Coot	Paul Emsley, LMB, Cambridge	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/	General software for model building but also available with other suites described above
DockinMap (Phenix)	Consortium of several institutes	https://phenix-online.org/documentation/reference/dock_in_map.html	Software inside the Phenix suite for docking model into cryoEM maps
Electron Microscopy Data Bank	Consortium of several institutes	https://www.ebi.ac.uk/emdb/	Public Repository for Electron Microscopy maps
Falcon	Thermo Fisher Scientific	https://assets.thermofisher.com/TFS-Assets/MSD/Technical-Notes/Falcon-3EC-Datasheet.pdf	Commercial, camera from Thermo Fisher
Phenix	Consortium of several institutes	https://phenix-online.org/download	Free for academic users and includes Coot
Protein Data Bank	Consortium of several institutes	https://rcsb.org	Public database of macromolecular structures
Pymol	Schrodinger	https://pymol.org/2/	Molecular viusalization tool. Educational version is free but comes with limitation. The full version can be obtained with a small fee.
Relion	MRC-LMB, Cambridge	https://relion.readthedocs.io/en/release-4.0/Installation.html	Software for cryoEM image processing, also available with CCP-EM
Titan Krios	Thermo Fisher Scientific	https://www.thermofisher.com/in/en/home/electron-microscopy/products/transmission-electron-microscopes/krios-g4-cryo-tem.html?cid=msd_ls_xbu_xmkt_tem-krios_285811_gl_pso_gaw_tpne1c& gad_source=1&gclid=CjwKCAiA-P-rBhBEEiwAQEXhHyw5c8MKThmdA AkZesWC4FYQSwIQRk ZApkj08MfYG040DtiiuL8 RihoCebEQAvD_BwE	Commercial, cryoTEM from Thermo Fisher
UCSF Chimera	UCSF, USA	https://www.cgl.ucsf.edu/chimera/download.html	General purpose software for display, analysis and more
UCSF Chimera X	UCSF, USA	https://www.cgl.ucsf.edu/chimerax/	General purpose software for display, analysis and more