BCH3052 Lecture Notes - Lecture 23: Crystallography, Small-Angle X-Ray Scattering, Eukaryote
25 May 2018
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Lecture 23 – Structural Dissection of the Bacterial Injectisome
• NMR and Crystallography limit proteins to be isolated
• Proteins rarely function alone
o Proteins localise to cytoplasm
• Injectisome
o Bacterial secretion system
o Approx 30 nm in diameter
o Functions: weapon as bacteria
▪ Injects bacteria in eukaryote cells
• Splicesome (from eukaryotics)
o Process eukaryote genes
o Composed of 5 small nuclear RNAs
• Has to be flexible
o Exist in different function states
• Use Electron Microscopy (EM) to visualise large complexes
o But at low resolution and expensive
o Have to use crystallography or NMR for higher resolution
▪ NMR can only be small molecules
• Lower resolution than crystallography:
▪ Not everything crystallises for crystallography
o The larger the molecular it is → the better the signal to NOS ratio
• Solve high resolution structure of components of proteins and dock them into
a lower resolution shape
How Different Levels of Structural Information can be integrated to Study Complex
Macromolecular Assemblies
Level of Structure
Method
3D atomic structure
Crystallography, NMR, homology modelling
Shape and Symmetry
Crystallography, NMR, homology modelling +
SAXS, EM
Protein contact (interaction)
Crystallography, NMR, EM + Chemical cross
linking, affinity purification/mass spec
Site directed mutagenesis, yeast two hybrid screen
SPR, ITC, computational docking
Subunit proximity (distances)
Crystallography, NMR + FRET, chemical cross
linking
The Basic Principles and Value of the Hybrid Approach using the Bacterial
Injectisome as an Example
• (Refer to cheat sheet) Crystallography – can only obtain atomic resolution
structures (most ideal)
• NMR spectroscopy
• Chemical cross-linking and FRET
o To determine distances
o Small molecular with two functional groups that react (amide, lysines)
• Advantage of hybrid (integrative) structural biology approach over a single
technique
o RACE: Resolution, Accuracy, Completeness, Efficiency
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