The neurobiologist's guide to structural biology: a primer on why macromolecular structure matters and how to evaluate structural data

Abstract

Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD).

Figure 1. Idea to Structure Flowchart

Basic steps of the process in solving a protein X-ray crystal structure are shown. Gray arrows show processes that are often iterated. (Step A) Optimization of constructs following outcome of expression tests. (Step B) Test of different expression hosts. (Step C) Crystal growth screening. (Step D) Crystal growth optimization. (Step E) Construct optimization to improve crystals. (Steps F and F1) Construct optimization to improve diffraction; (step F2), search for new crystallization or cryoprotectant conditions; (step F3) heavy atom soaks. (Step G) Production of selenomethionine-labeled protein for MAD and SAD experiments. (Step H) Building and refinement cycle for refining structure.

Figure 2. Anatomy of a Protein Crystal

(A) Example of an asymmetric unit (AChBP). In this case the asymmetric unit is the same as the biological unit. (B) Crystallographic symmetry operators applied to the asymmetric unit create the unit cell. Note that the AChBP pentamers in the upper right and lower left corners of the cell are in the opposite orientation of the orange pentamer. (C) Translations of the unit cell build the protein crystal lattice.

Figure 3. Diffraction Experiment Schematic

(A) Standard setup for data collection. The protein crystal is mounted on a goniometer (orange) and is frozen in a nylon loop. Incoming nitrogen gas at 100K maintains the frozen state of the crystal. X-rays emerge from a collimator on the X-ray source. (B) Diffracted X-rays are collected on a detector. An example of an X-ray diffraction pattern is shown with low- and high-resolution data regions indicated. In the actual setup, the detector face is normal to the X-rays. (C) Example of an initial electron density map at 2.0 Å with phases determined by selenomethionine MAD. A helix can be seen prominently on the right-hand side. (D) Final refined model.

Figure 4. Example of SAXS Analysis

(A) Top shows the ab initio model for SAXS data from the Kv4.3 T1 domain/KChIP1 complex calculated from the scattering data. The bottom panel shows the scattering intensity profile for the data (black) and the model (red). (B) Comparison of the crystal structure of the Kv4.3 T1 domain/KChIP1 complex with the ab initio model shows excellent correlation with the data in contrast to an alternative square-shaped arrangement shown in (C). The D_max values show the maximal dimension of the particles calculated from the data (A) and from the models (B and C). Data are from Pioletti et al. (2006).