Structural biology methods

Why it matters

Knowing a protein's atomic structure lets you see how an enzyme holds its substrate, where a drug binds, how a mutation causes disease and how to engineer a better antibody. Three experimental methods produce almost every structure in the Protein Data Bank: X-ray crystallography, cryo-electron microscopy (cryo-EM) and NMR. Since about 2015 cryo-EM has grown dramatically, and AI prediction (next lesson) changed the field again, so knowing what each method needs, delivers and misses is essential for reading papers and planning projects.

X-ray crystallography

Idea. Grow a well-ordered crystal of the protein, shine an intense X-ray beam (often at a synchrotron) through it, and record the diffraction pattern. The spots' positions come from the crystal lattice (Bragg's law, nλ = 2d·sinθ), and their intensities encode the electron density in each repeating unit.

The phase problem. The detector records intensities but not the phases of the diffracted waves, which are needed to compute the electron density. Phases come from molecular replacement (using a similar known structure or a predicted model, now the most common route), or experimentally from heavy atoms or anomalous scatterers such as selenomethionine.

Then: build an atomic model into the density map and refine it. Quality indicators include resolution, R-work and R-free (agreement with data held out from refinement; a large gap from R-work warns of overfitting).

Strengths: very high resolution is possible (better than 1.5 Å, showing waters and alternative side-chain positions); mature, fast pipelines for ligand soaking in drug discovery; works for small proteins.

Weaknesses: you need crystals, which can take months or never form; flexible regions are often missing; crystal packing can distort surfaces; one static snapshot.

Cryo-electron microscopy (single particle)

Idea. Flash-freeze a thin film of the protein solution so fast that water forms glass-like vitreous ice, preserving molecules in near-native states. Image thousands to millions of individual particles in an electron microscope at very low dose (electrons damage proteins), then use software to classify and average 2D images into a 3D map.

Key advances behind the "resolution revolution" (Nobel Prize in Chemistry 2017): direct electron detectors, motion correction and much better image-processing algorithms.

Strengths: no crystals needed; excellent for large complexes and membrane proteins (ribosomes, ion channels, viral spikes); can separate several conformations in one sample; needs relatively little protein.

Weaknesses: small proteins (below roughly 50–100 kDa) are hard because they give little contrast; resolution is often 2.5–4 Å, so side chains and ligands may be less certain; microscopes cost millions and data sets are huge; sample preparation (ice thickness, particles sticking to the air–water interface) is still a craft.

Cryo-electron tomography goes further, imaging molecules inside thin slices of cells, at lower resolution.

NMR (brief)

NMR determines structures in solution from distance and angle restraints, and it uniquely captures dynamics. It is limited to smaller proteins (typically below about 30–40 kDa) and needs isotope labelling.

Comparison

                   X-ray                 Cryo-EM                  NMR
sample             crystals              frozen solution          concentrated labelled solution
best size range    any (if it crystallises)  large (> ~100 kDa)   small (< ~30–40 kDa)
typical resolution 1–3 Å                 2–4 Å                    restraint ensemble
dynamics           limited               several states           yes, residue level
main bottleneck    crystallisation       sample prep, contrast    size, labelling

Worked example: choosing a method

1. A 25 kDa soluble enzyme, crystals already grow, and you want to see 50 drug candidates bound. X-ray: crystal soaking gives high-resolution ligand poses quickly.
2. A 400 kDa membrane transporter that will not crystallise. Cryo-EM, perhaps in detergent micelles or nanodiscs.
3. A 12 kDa domain with a flexible tail that changes shape when it binds a partner. NMR, possibly combined with an AlphaFold model.

Reading a structure paper critically

• Check resolution and, for X-ray, R-free; for cryo-EM, look at the local resolution map: the core may be 2.8 Å while flexible edges are 5 Å.
• Is the ligand's density convincing, or is the ligand modelled into a weak blob?
• What construct was used? Truncations, stabilising mutations, fusion partners and antibodies used to help crystallisation or particle alignment can change the picture.
• Are conclusions about motion supported by more than one static structure?

Safety and access

Synchrotron and microscope time is booked through facilities with mandatory radiation, cryogen (liquid nitrogen and ethane) and equipment safety training. You use these instruments only after that training and under facility supervision.

Recap

• X-ray: crystals, diffraction, the phase problem, potentially the highest resolution.
• Cryo-EM: vitreous ice, single-particle averaging, best for large complexes and multiple states.
• NMR: small proteins in solution, with dynamics.
• Match the method to the protein's size, flexibility and the question.

Try it

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