crystallography and NMR Flashcards
explain the basis of crystallography
pure sample→protein crystal
highly purified protein sample is put in crystallisation conditions to make it rearrange into an ordered crystal lattice
protein crystal→diffraction pattern
crystal is exposed to xrays, when xrays hit crystal they scatter in specific directions creating a diffraction pattern, the pattern contains info about electron density within the crystal
diffraction pattern→structure
computer analyses the diffraction pattern to reconstruct an electron density map of protein, the atomic model of protein is fit in, high resolution 3D structure of the protein is produced, shows info about its shape, function and interactions
explain protein electron density maps, what does the detail depend on
-xray diffraction gives an electron density MAP not structure
-detail of map depends on quality of crystal
-expressed as the resolution of the map in angstroms
what does electron density resolution value indicate
how accurately the drug structure is described which is important for drug design (reflects xray experiment)
how to obtain an inhibitor target complex structure (to see how inhibitor binds to active site of a crystallised enzyme)
- soak inhibitor in preformed crystals
- co crystallisation
-grow a native crystal of the protein then change the liquid around the crystal, incubate for a certain amount of time with drug, crystals have solvent channels penetrating through, right conditions then drug can access enzyme active site and form drug complex
how to tell if an inhibitor has bound in crystal soaking experiment
-measure diffraction pattern of native and drug complex
-changes in intensity
how to find isomorphous differences
- collect native dataset (Fp)
- introduce inhibitor
- collect derivative dataset (Fph)
- solve the difference structure (Fh) (drug complex-native=Fh)
assumptions for crystal soaking experiment
-drug must be compatible with the mother liquor
-drug binds to protein in the crystal (binding site is exposed)
-crystal must be isomorphous (no changes in unit cell dimension)
what does isomorphous mean
two crystals share same space group and unit cell dimensions, eg. crystal lattice remains unchanged, if ligand binding distorts unit cell then non isomorphous (?)
advantages and disadvantages of protein crystallography
advantages
-experimental phase, hard to get false positive as xray doesnt lie, high confidence
disadvantages
-lots of work to generate crystals, needs generate lots of proteins and do a lot of screens, crystals dont always diffract, crystals not always amenable to soaking, important to get high resolution to get correct bound orientation
2 methods of designing lead compounds using NMR spec
ligand or protein observed
in ligand observed screening, what does STD stand for
saturation transfer difference
explain ligand observed STD NMR spec (what is it influenced by, what can it do, when do NMR signals appear,used for what, detects what)
-NMR properties influenced by molecular size
-can differentiate between binders and nonbinders in a mixture of fragments
-focus on changes in ligand
-NMR signals when ligand binds to protein
-used to screen weak binders
-detects binding
what is HSQC in protein observed screening
heteronuclear single quantum coherence
explain protein observed screening (what does it do, use and detects what)
-fragment binding by HSQC
-focuses on chemical shifts in signals when ligand binds
-used for mapping binding sites and studying conformational changes
-detects binding site and interactions
what are protein observed inhibitor binding sites
hydrophobic grooves, location varies based on HSQC data, contact with domain interface, consistent with xray data
requirements for NMR protein samples
-need to be ~10mg of protein
-pure, water soluble
-doesnt have to form crystals
-weight limit of 40 kilodaltons/350 amino acids
-isotopically enriched
basis of NMR spec
protein solution in spectrometer to make NMR spectra then through computer to get HH distances and bond torsion angles then computer predicts structure
2D NMR spec gives info on what
secondary and tertiary protein structure
uses of NMR spec in drug discovery
-structure determination
-HSQC solution binding to inhibitors
-STD NMR medium throughput screening
-screening of drug fragments
-SAR by NMR (structure activity relationship)
draw the design of lead compounds using NMR spectroscopy
notes
why is NMR based structural determination challenging
-large quantities of very pure/soluble protein needed
-weight limit of ~350 amino acids
-difficult to analyse spectra due to overlapping peaks
-only info on short range structure
-parts of structure may be blurred due to lack of data or dynamics
why is xray crystallography challenging
-not all proteins form crystals
-not all crystals diffract xray
-not all diffraction patterns can be solved (converted into electron density maps)
-not all electron density maps can be interpreted (converted into structures)
