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Protein Solving
Uni of Notts, Structure, function, & analysis of Proteins, year 2, topics 11&16
| Term | Definition |
|---|---|
| X‑ray crystallography | Structural method that observes X‑ray diffraction patterns from protein crystals to map electron density |
| Bragg’s law | The equation nλ = d·sinθ describing how X‑rays diffract from crystal planes |
| Phase determination | Solving missing phase information (from phase shifts in the X-ray beams) by adding heavy atoms (e.g., Se, W*) to locate electron density |
| Crystallography strengths & weaknesses | STR: High atomic detail, automated workflows, most productive structural method WKN: Needs many pure crystals, soluble proteins only, no dynamics, packing artefacts, radiation damage |
| NMR spectroscopy | Aligns magnetic nuclei, perturbs them with radio pulses, & realigns to measure through-bond & through-space distances by measuring unabsorbed RF |
| NMR structure indicators | Produces a spectra showing fingerprint regions for functional groups & 2* structures. α‑helices show HN (N-H+)–HN (i/i+1) or HN–HA (Cα-H+) (i/i+3); β‑sheets show HA–HN (i+1) |
| NMR workflow | Purify protein, collect spectra, derive constraints, compute full atomic structure |
| NMR strengths & weaknesses | STR: Determines full structure, works in native solution, reveals dynamics WKN: Works best <20 kDa; sometimes 40–80 kDa; often requires isotopic labelling |
| Cryo‑EM (+vitrified water) | Electron microscopy using vitrified water (Rapid freezing of water into a glass‑like state without crystals) to preserve proteins in native conformations |
| Cryo‑EM strengths & weaknesses | STR: Determines full structures, all proteins, no staining/crystals, ideal for large complexes WKN: Poor resolution for <100 kDa; struggles with symmetric heteromers & protein polymorphisms |
| Hydrodynamic methods | Ultracentrifugation (10⁵–10⁶ g) with Schlieren (S) optical detection to estimate protein size from sedimentation velocity (smaller proteins sediment slower & diffuse faster) using light beams & fluorescence |
| Spectroscopic methods | Circular dichrosim uses enantiomers to rotate plane-polarised light. Raman is relative frequencies samples scatter photons. Infrared is absolute frequencies samples absorb |
| In‑silico modelling | Predicts structure using templates (related proteins), template‑free methods (hypothetical based on sequence & similarities), or fragment libraries (comparing 3-15AA residue stretches to known proteins) |
| AI deep‑learning folding | In-silico AI (e.g., AlphaFold) predicts 3D structure from sequence using large datasets |
| Membrane‑protein under‑representation *example* | Membrane proteins are ~25% of genes but ~100× under‑represented in PDB |
| EM advancements | Lenses, detectors, & noise reduction improved resolution from ~6 Å to near‑atomic as well as Charge Coupled Devices (multiply reflected e- for stronger signal) |
| Quantum efficiency | How effectively detectors convert incoming particles (like photons or electrons) into a measurable signal |
| Single‑particle imaging | Millions of particle images are categorised, aligned, averaged, & reconstructed into 3D |
| Bacteriarhodopsin in studying membrane proteins | Light‑driven proton pump using retinal isomerisation to power ATP synthase. Extremely abundant membrane protein; cryo‑EM can freeze membranes to capture fixed conformations |
| 2D cryo-EM | Cryo‑EM of membrane‑embedded proteins in identical orientations to view top‑down structure |
Created by:
Denny12
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