NMR 1 Flashcards
1
Q
What are some Experimental methods that permit determination of biomolecular structures at atomic resolution
A
- X-ray crystallography (XRC)
- Solution state nuclear magnetic resonance (NMR) spectroscopy
- Single particle cryo-electron microscopy (cryo-EM)
- Solid state NMR
2
Q
What is the niche of NMR in structural biology
A
- NMR can be used to determine structures of proteins and complexes
- Strength of NMR derives from
- its capacity to study proteins at level of individual amino acids,
- and exquisite sensitivity of magnetic nuclei to changes in chemical environment
3
Q
Approximate molecular mass limitations for atomic resolution structure determination
A
- XRC: not limited by molecular mass, in principle
- Solution NMR: < 60 kDa
- Cryo-EM: > 50-100 kDa
- Solid state NMR: not limited by molecular mass, in principle, but signal number and overlap constitute significant practical obstacles
4
Q
What are some characteristics of XRC
A
- > 90% of structures in PDB
- Crystals of molecule/assembly needed
- Difficulty of crystallisation increases with mass and complexity of target
- Only about 23% of XRC structures in PDB exceed 100 kDa, whereas 60-80% of proteins oligomerise
- Some proteins and assemblies are too flexible/amorphous, adopting multiple conformations, to crystallise
5
Q
What are some characteristics of Solution NMR
A
- Crystals not needed
- Study molecules in aqueous solution or inside cells
- Molecule/assembly isotope labelled (typically one or more of 15N, 13C, 2H)
- Together with cryo-EM, has contributed to about 10% of structures in PDB
- Size limit (fast tumbling) for atomic res. structure determination
- Width of peaks or signals Is controlled by tumbling
- Bigger molecules tumble more slowly
- “Tricks” such as TROSY and deuteration allow study of larger (< 500 kDa) systems
- Methyl-TROSY extends limit to 1000 kDa
- Several applications beyond structure determination (e.g. binding, folding, dynamics)
6
Q
What are some characteristics of Cryo-EM
A
- Molecule/assembly studied in frozen state within thin film of amorphous ice
- Single particle analysis to reconstruct 3D electron density map
- “Resolution revolution” (direct electron detectors, image processing)
- Cryo-electron tomography to visualise molecule/assembly in cell
- Mol wt range ~100 kDa to > 11 MDa
- Conformational heterogeneity before freezing can limit resolution
7
Q
What are some characteristics of Solid state NMR (ssNMR)
A
- Molecule/assembly does not need to be soluble or crystal
- Protein/assembly isotope labelled (typically one or more of 15N, 13C, 2H)
- Dry samples, hydrated solids, sedimented soluble assemblies etc.
- Low sensitivity and spectral crowding
8
Q
What methods are used for Awkward squad” proteins
A
- integrated structural biology approach
- Not amenable to crystallisation
- Produce poor NMR spectra
- Too conformationally heterogenous for cryo-EM
- Recent approach - integrate data from multiple biophysical methods to determine a structural model:
- integrated structural biology or integrative modelling
9
Q
What are some complementary low resolution biophysical methods
A
- Atomic force microscopy (AFM)
- Cross-linking
- Förster resonance energy transfer (FRET)
- Light scattering e.g. size exclusion chromatography-multi-angle light scattering (SEC-MALS)
- Mass spectrometry (e.g. HDX), proteomics
- Small angle X-ray or neutron scattering (SAXS or SANS)
- CD spectroscopy (CD)
10
Q
What is the physical basis of NMR
A
- Some nuclei have a magnetic dipole – they are magnetic, rather like a bar magnet.
- Without an external magnetic field, there is no energy difference between different orientations of nuclear magnets – the distribution of magnetic moments is isotropic.
- Magnetic nuclei have non-zero spin quantum numbers
11
Q
What is spin quantum number for different nuclei
A
- Nuclei with odd mass numbers have half-integer spin quantum numbers
a) e.g. 13C, 1H, 31P: I = 1/2
b) 17O: I = 5/2 - Nuclei with an even mass number and an even charge number have spin quantum numbers of zero
a) e.g. 12C: I = 0 - Nuclei with an even mass number and an odd charge number have integer spin quantum numbers
a) e.g. 2H: I = 1 - Electrons also have a spin quantum number of 1/2
12
Q
Describe effect of external magnetic field on nuclei
A
- In an external magnetic field, usually labelled B or B0, certain orientations of magnetic nuclei are preferred.
- A magnetic nucleus oriented with (parallel to) the external field has lower energy than a nucleus oriented against (antiparallel to) the field.
- Placing nuclei with spin I = 1/2 into a magnetic field leads to a net magnetisation aligned along the magnetic field axis
- Parallel = +1/2
- Antiparallel= -1/2
13
Q
How are NMR signals produced
A
- In an external magnetic field, nuclear magnets adopt different orientations, each having a different energy.
- Applying pulses of electromagnetic radiation at frequencies that precisely match the energy gaps allows observation of transitions that produce NMR signals.
14
Q
Describe an NMR magnet
A
- Big steel can
- Ports at top
- Can contain liquid nitrogen and helium
- Ports allow these to get into can
- Material used to generate magnetic field is super conducting
- Once it is energised it conducts electricity without resistance
- Used at very low temperatures – liquid helium
- So retains superconducting properties
- Liquid helium chamber is insulated with liquid nitrogen chamber to lower cost and keep safe
15
Q
How are proteins detected in NMR
A
- Usually, observe protons (1H)
- Different from X-ray diffraction structure determination which is based on the electron density from electron-rich atoms (C, N, O)
- Protein dissolved in water
- Protons have spin angular momentum (“spin” for short)
- Protons behave like small bar magnets and align with or against a magnetic field
- These small magnets interact with each other
