Modeling, docking, and fitting of atomic structures to 3D maps from cryo-electron microscopy

Abstract

Electron microscopy (EM) and image analysis offer an effective approach for determining the three-dimensional structure of macromolecular complexes. The versatility of these methods means that molecular species not normally amenable to other structural methods, e.g., X-ray crystallography and NMR spectroscopy, can be analyzed. However, the resolution of EM structures is often too low to provide an atomic model directly by chain tracing. Instead, a combination of modeling and fitting can be an effective way to analyze the EM structure at an atomic level, thus allowing localization of subunits or evaluation of conformational changes. Here we describe the steps involved in this process: building a homology model, fitting this model to an EM map, and using computational methods for docking of additional domains to the model. As an example, we illustrate the methods using an integral membrane protein, CopA, which functions to pump copper across the membrane in an ATP-dependent manner. In this example, we build a homology model based on the published atomic coordinates for a related calcium pump from sarcoplasmic reticulum (SERCA). After fitting this homology model to a 17 Å resolution EM map, computational software is used to dock a metal-binding domain (MBD) that is unique to the copper pump. Although this software identifies a number of plausible interfaces for docking, the constraints of the EM map steer us to select a unique solution. Thus, the synergy of these two methods allows us to describe both the location of the unknown MBD relative to the other cytoplasmic domains and the atomic details of the domain interface.

Figure 1

Topology of the proteins used for modeling. (A) The Ca²⁺-ATPase from sarcoplasmic reticulum (SERCA) provided the template for homology modeling. SERCA consists of ten transmembrane domains and two large cytoplasmic loops. In the 3D structure of SERCA, these cytoplasmic loops form three domains, which are denoted A-, N-, and P-domains. (B). The Cu-ATPase from Archeaoglobus fulgidis (CopA) has eight transmembrane helices. Six of these helices and the two cytoplasmic loops are analogous to the catalytic core of the SERCA molecule. Specialized metal binding domains with the conserved CxxC motif are present on both the N-terminus (NMBD) and C-terminus (CMBD). Two additional transmembrane helices are also associated with the N-terminus of CopA.

Figure 2

Selecting the appropriate conformation for homology modeling. In the case of SERCA, the various different X-ray crystal structures show that the linker between the N- and P-domains is flexible. (A) The cytoplasmic domains from the EM density map of CopA at 17 Å resolution (1). The locations of the A-, N-, and P-domains are indicated. (B) After superimposing the P-domains from three atomic structures from SERCA, their N-domains are seen to adopt different positions. The position of the N-domain in the red model, which corresponds to 1IWO, is most consistent with the EM density map. PDB codes for the three structures are 1IWO (red), 1SUH (pink), 1VFP (olive). The corresponding A-domains have been omitted for clarity.

Figure 3

Homology model for CopA. (A) The crystal structure of SERCA in the E2 conformation (1IWO) provided the template for building the homology model for CopA. SERCA does not have an NMBD, so a separate template (inset) was used, namely a copper metallochaperone (2QIF) which has high sequence homology with NMBD. (B) The resulting homology models were produced by ICM. Note that CopA lacks a number of loops in the three cytoplasmic domains but that the overall configuration of these domains is preserved. Some of the transmembrane helices appear to have been misfolded and will need manual rebuilding for optimal fitting to the map. Domains have been colored as follows: N-domain green, P-domain yellow, A-domain red, transmembrane domain blue, metal binding domain purple. SERCA also has two N-terminal helices associated with its A-domain (light blue).

Figure 4

Docking Results in ICM. The graphical user interface for ICM has several windows for viewing relevant information. The main display window allows interactive visualization of the model. The top-left window allows selection of objects for viewing in the main display window. The bottom-left window shows a table of docking results sorted by energy value; clicking an individual row displays that solution in the visualization window. The bottom-right window displays a plot of each docking solution versus energy (on the abscissa). Low energy solutions (e.g., red square) that are well separated from the remainder indicate a successful docking experiment.

Figure 5

Result of fitting the model into the EM map. (A) Side view of the cytoplasmic domains from the final model fitted into the 17 Å resolution EM map. (B) Top view of these cytoplasmic domains, obtained after a 90° rotation about the horizontal axis. The oblique black line represents the boundary with a two-fold related molecule that has not been fitted with a corresponding model. This model represents the lowest energy structure obtained from the docking experiment and appears to produce an optimal fit with the map.