Accelerator Mass Spectrometer Schematic For Radiocarbon
6.1 Shape, secondary structure, and dynamics in bulk #
6.1.1 Small-angle X-ray scattering #
SAXS records low-angle scattering from a macromolecule in solution, averaged over orientations and over every particle in the beam. It does not conceal an atomic model. A Guinier plot gives the radius of gyration. The pair-distance distribution gives Dmax, the longest dimension. A Kratky plot separates a compact fold from an unfolded chain. Bead envelopes, at roughly one to two nanometres, are not unique. Safe uses are oligomeric state, dimensions, and a test of whether a rigid model matches the solution particle. Forward scattering, scaled by concentration, grows with mass, so a trace of aggregate dominates. An Rg that drifts with concentration is a warning.
6.1.2 Circular dichroism #
Far-ultraviolet circular dichroism reports secondary-structure content. A helix has negative bands near 208 and 222 nm; a β sheet near 215–218 nm; disorder near 200 nm. Percentages are approximate and do not say which residues are helical. Near-ultraviolet CD is a tertiary fingerprint of aromatics and disulphides, not a structure. The proper use is comparison: whether a mutant is still folded, or whether a condition melts the helix. A melting temperature is not a free energy. “CD confirmed the structure” means only that the content, or the fingerprint, matched expectation.
6.1.3 Hydrogen–deuterium exchange #
Backbone amides exchange with solvent. Buried, hydrogen-bonded amides exchange slowly; exposed free amides exchange quickly. In deuterated water the mass change is read by mass spectrometry, peptide by peptide, or by NMR, residue by residue. Exchange depends strongly on pH and temperature, which is why it can be quenched by lowering both before analysis. That is the principle, not a protocol. Protection after ligand binding may be the contact, or an allosteric tightening. The experiment does not decide, and it does not produce a fold. Neighbouring peptides can differ by orders of magnitude. Flexibility is not uniform.
6.2 Distances and cellular context #
6.2.1 Cross-linking mass spectrometry #
A cross-link joins side chains within reach of a reagent of known chemistry, often an NHS ester aimed at lysine. Mass spectrometry identifies the peptides. The result is an upper bound: once side chains and linker flexibility are included, typically a few tens of ångströms between α-carbons, not a van der Waals contact and not the spacer length on the bottle. Links are sparse, biased to reactive surface residues, and true at some moment during the reaction. In an oligomer they may be within or between chains. A set can arrange known subunits or falsify an arrangement. One link drawn as a salt bridge is an overreading.
6.2.2 Super-resolution microscopy and atomic force microscopy #
Visible light is diffraction-limited at about 200 nm. STED, and single-molecule methods such as PALM and STORM, localise fluorophores to some tens of nanometres. The optics are Biophysical Techniques. The structural limit is the claim: these methods place a label in a cell. They do not show side chains, and the coordinate is the dye or antibody, which has a size. Atomic force microscopy records surface height. Lateral resolution is typically a few nanometres and is tip-limited; the interior is invisible. In liquid it can follow an exposed conformational cycle. A force of unfolding is not the thermodynamic stability of Chapter 2.
6.3 Choosing #
6.3.1 Question, method, and limit #
| Question | Method | It can say | It cannot say |
|---|---|---|---|
| Atomic model, including ordered waters, of a domain that crystallises | X-ray crystallography | Side chains and ligands at the resolution achieved | That this conformer is the only one in solution |
| Dynamics, disorder, or a weak complex under about 40 kDa | Solution NMR | Ensemble, timescale, solution surface | A first atomic model of a megadalton asymmetric particle |
| Near-atomic model of a large complex or membrane protein | Single-particle cryo-EM | A map and, locally, a chain trace | One structure if the sample is a continuum |
| Oligomeric state and domain arrangement in solution | SAXS | Rg, Dmax, fit to a rigid model | A unique atomic model, especially if aggregates remain |
| Is the mutant folded, and roughly how helical? | Far-UV CD | A change in secondary-structure content | Which residues |
| Which peptides are protected on binding? | Hydrogen–deuterium exchange | Regional protection compared between states | A fold, or a guaranteed contact |
| Which subunits lie within tens of ångströms? | Cross-linking MS | Sparse upper bounds | A named hydrogen bond |
| Where is the assembly in the cell? | Super-resolution fluorescence | Position of the label | An atomic model |
| What does the surface do in a bilayer? | AFM | A nanometre-scale contour | The buried interior |
The right-hand column is part of the answer.
6.3.2 Hybrid models #
A large assembly is often a hybrid: domain structures, a cryo-EM map, cross-links, a SAXS curve, sometimes a tomogram. That is legitimate when no single method sees every part. Each restraint should be shown to have changed the model or to have been satisfied, and unrestrained parts should look different from a 2 Å decision. A figure in which a guessed loop and a crystallographic side chain are painted the same is an illustration. Integrative archive entries exist so the bookkeeping can be deposited.
6.4 Key terms, summary, and self-check #
Key terms. SAXS; radius of gyration; circular dichroism; hydrogen–deuterium exchange; cross-link; super-resolution microscopy; atomic force microscopy; hybrid model.
Summary. SAXS gives solution shape and is easily dominated by aggregates. Circular dichroism gives secondary-structure content, not a fold. Hydrogen exchange reports protection, not the reason for it. Cross-links are sparse upper bounds of tens of ångströms. Super-resolution microscopy and AFM locate or contour assemblies and do not give atomic models. The choice of method is a choice of limits. A hybrid model is valid only while those limits stay visible.
Self-check.
1. What does a Guinier plot give, and why can a trace of aggregate dominate SAXS?
2. Far-UV CD says a protein is about 40% helix. What is still unknown?
3. A peptide is more protected after ligand binding. Give a local and an allosteric reading.
4. Why is a lysine cross-link an upper bound of tens of ångströms, not a contact?
5. Which method places a complex in the cell, and why does that not compete with a cryo-EM map?
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