1.3 Structural methods for macromolecules

Question 1

What are the main macromolecular structure methods?

Answer 1

• X-ray diffraction
• Cryo-EM
• NMR

Question 2

How are protein samples prepared for structural analysis?

Answer 2

Relatively large protein sample needed - clone and express proetin in bacterial host:
- specific restriction enzymes cut particular needed gene
- same restriction enzyme used to cut bacterial vector (plasmid)
- the gene ligated into the vector (+ can also be engineered to add tags to targeted proteins ex: 6 histidine tag - for purification)
- vector transformed into bacteria
- bacteria cultured
- protein purified from bacteria by cell lysis + chromatography

Question 3

How engineered 6 histidine tag helps in protein purification?

Answer 3

6 histidine tag: histidines have lone pairs (N:) which can bond to transition metals

Question 4

Explain how 6 histidine tagged proteins are purified in chromatography

Answer 4

Contaminated sample with other proteins
- 6 his tagged proteins bind to Ni2+ in Ni-NTA column - contaminant proteins washed out
- 6 his tagged proteins eluted using imidazole - binds to Ni2+ instead of 6 his (competition for binding)
- 6 his tag removed by washing the tagged protein through the same column again

Question 5

Explain how X-ray crystallography works

Answer 5

Sequence of events in X-ray crystallography:
- protein purification -> protein sample
- protein crystals formed (making proteins less soluble)
- crystal diffraction patterns - ordered lattices provide structured patterns
- diffraction patterns fourier transformed - electron density analysed (electron density map) - molecule models constructed (using computers)
- Refinement - checking if this structure is possible -> phases (mathematical consideration) -> fourier transform (cycle repeated several times to construct most correct structure)

Question 6

Which molecules is X-ray crystallography used for? What are the limitations of this method?

Answer 6

Can be used for small / big molecules EXCEPT:
- quarternary proteins (hard to crystalise)
- membrane bound proteins (hard to find hydrophob. solvent)

Question 7

Is higher or lower resolution better to identify molecules in X-ray crystallography?

Answer 7

Higher resolution (Å lower) - different size atoms can be distinguished

Question 8

Explain how Cryo-EM works

Answer 8

Cryo-EM - direct visualisation (molecular microscopy):
- samples (very pure samples needed) frozen on thin carbon film in liquid nitroge
- Cryo-EM observation through microscope
- digital microgrpahs produced 2D - to construct 3D model many micrographs from different angles needed - image alignmnet
- computers align mutiple micrographs and construct 3D model

Question 9

Which molecules is Cryo-EM used for? What are the limitations of this method?

Answer 9

Good at visualising large molecules (ex: viruses)
Limitation: protein samples must be very pure, not ideal for small molecules

Question 10

Explain how NMR spectroscopy works

Answer 10

NMR spectroscopy:
- protein sample istopically labelled
- NMR spectroscopy
- Alignment (-> secondary structure)
- Calculations (conformational constraints, geometry) -> structure refinement (cycel repeated several times to construct most correct structure)

Question 11

Which molecules is NMR spectroscopy used for? What are the limitations of this method?

Answer 11

NMR spectroscopy best for small molecules
Limitations: pure sample needed, not ideal for large molecules