Karteikarten
Protein crystallisation 3
Karten in diesem Stapel (38)
- 1what are the 3 stages of building a structure from protein crystallography- Tracing the protein
- Refinement
- Structure validation - 2what is the tracing step of structure building- Trace the protein chain with the bones that are provided by the electron density map —>this is the main chain/protein backbone
- This is typically done automatically
- Main chain and that the side chains can then be added to
- Once the bones have been drawn, the protein main chain is determined And the atoms are added (this includes the protein side chains) - 3what is the refinement in structure building- To improve the phases (that have been obtained through the 3 solutions to the phase problem) and thus get a more accurate model
- Iterative cycles of manual correction and automated statistical adjustments —>looking at the electron density map and use own knowledge of the protein side chains —>10-20 cycles
- During these cycles you are adjusting atomic positions within the model (x,y,z) and thermal parameter (indication of movement) to fit experimental data
- High flexibility= high thermal parameter - 4what are the 2 different types of electro density map that are used when refining a structure- 2Fobs-Fcalc = ‘normal’ map
- Fobs-Fcalc = ‘difference’ map - 5what are the Fobs (refinement of a structural density map)Fobs are observed amplitudes
- 6what are the Fcalc values that are used to calculate the phase informationcalculated using the model that has been created
- 7what are the restraints used in refinement of an electron density map- These are rules that are known from chemistry e.g.
—>bond lengths
—>band angles
—>planarity
We know e.g. that in a peptide bond the lengths of bonds between certain atoms is constant
- This is not to say that every thing fits within these rules, there are occasions where bonds are different in length from expect
- This could be caused by difference in the active site for example binding
- Vast majority restraints apply - 8why are there restraints that are placed on the refinement of an electron density map- These are known from chemistry Of the structures
- Makes the structure more accurate
- Ensure that the structure meets certain parameters
- Improves the observation/parameter ratio - 9why is refinement of the structure measured- Refinement has to be monitored to ensure that the model is being improved and not causing any bias
- 10how is refinement measured-using the R value
This gives an indication of agreement between the model and the experimental data - 11how is the R value calculatedwhere F is the amplitude
this extends over all measurements of reflections - 12what should the R value be- A perfect model would have a R factor of 0- however this is reality never happens
- Typically values are around 0.15-0.25 (this is dependent on the resolution of the model)
- Higher resolution data result in a lower R factor, higher resolution that doesn’t fit as closely to experimental data will result in a higher R factor - 13what measurement can be used as a monitor of bias on the refinementR free value
- 14how is the R free calculated- Uses the same equation as the R-factor but for a subset of reflections not included in refinement
- Typically 5% of the reflections are not recorded in the refinement
- ‘A reality check’—> if we are causing bias the R factor will reduce but the R free will not - 15how are the R and R free values compared- Value of the R free always higher (typically want less than a 0.05 gap between the R factor and the R free)
- 16what is structure validation- Model refinement has subjective elements so needs checking for validity
- Check consistency of model against the experimental data —> haven’t overfitted the model and introduced bias
- Check the consistency of model against known physical and chemical parameters —>knowing the bond angles, length and planarity - 17what can be used in the validation of a structureramachandran plot
Looking at the main chain of a protein checking if it is to have Phi and Psi angles or torsional angles within the side chains can be plotted and outliers identified - 18what are chi anglesside chains can be assessed to ensure that the chi angles (torsional angles) within the side chains can be plotted and outliers are spotted
- 19what is the resolution of crystallography- Resolution is the distance corresponding to the smallest observable feature
- 1 angstrom id high resolution where the features would be well defined - 20what is the average resolution for most structures using current technology- This averages at around 2-2.3 angstrom for most structures
- The resolution is dependent on the quality of, and ordering of molecules, within the crystals —>if the crystal is not well ordered then they are not held or packed tightly
—>tight packing = high resolution - 21what can affect the resolution of crystals>can be effected by the volume of water that is in crystals
—>normal crystals have around 50% solvent, high resolution data is typically derived from crystals that have a lower solvent concentration as this means crystals are able to pack tighter - 22what are the two ways in which it is possible to obtain a structure of a protein interaction with a ligand- Soak the crystals with a small molecule inhibitor
- Co- crystallise the protein with the ligand - 23what are the disadvantages to soaking crystals with a small molecule inhibitor for the identification of protein-substrate interaction- Soaking can cause the protein crystal to fracture and reduce diffraction
- 24what are the disadvantages to Co- crystallising the protein with the ligand for observation go the protein substrate interaction- Co-crystallisation and the presence of small molecules can mean that crystals do not form under the same conditions as the regular protein
- 25what interactions may it be possible to see in the active site of an enzyme that is complexed with an enzymeHydrogen bonds
Hydrophobic interactions
Water mediated interactions
Charge charge electro static interactions
Pi-pi stacking interactions - 26looking at hydrogen bond interactions in the active site of an enzyme using crystallography- Hydrogen bonds typically range between 2.4 and 3.2 A
- Closer than this is usually a mistake as the van der waals would not allow this
- Polar interactions between hydroxyl groups and groups such as carboxylate or nitrogen in residues
- 27looking at hydrophobic interactions interactions in the active site of an enzyme using crystallography- Interactions between hydrophobic residues such as phenylalanine
- 28looking at water mediated interactions interactions in the active site of an enzyme using crystallography- There are often water molecules within the structure and these can typically mediate reactions between the inhibitor and the active site
- Although they do not bond directly they are important to look at in complexes that involve bonding to active sites
- 29looking at charge charge interactions interactions in the active site of an enzyme using crystallography- Carboxylate group (-) and Arginine (+)
- Pos and neg cause strong interactions
- 30looking at Pi pi stacking interactions interactions in the active site of an enzyme using crystallographyWhere there is a delocalised planar phenylalanine causing a stacking
- 31why can understanding interactions in binding sites can be useful in the development of drugs- Able to determine the pocket shapes of the active site allowing for design (what resides and side chains??) of drugs and inhibitors that fit with tight affinity into the binding site
- 32Give an example of a drug that has been developed using structure based drug design- Relenza was developed using structure based drug design
- 33what can make working with membrane protein difficultHard to work with as they are the opposite of many proteins- with a hydrophilic core and a hydrophobic exterior
- Do not like to be purified in aqueous buffer- lipid typically have to be added - 34how has crystallography been used to understand enzyme mechanisms- Lysozyme is a common protein and the first enzyme structure that was solved
- Believe for 50+ years that is used a long lived non covalent intermediate
- Was used as a typical and classic example in many textbooks
- However structural studies using a synthetic substrate trapping the covalent intermediate —>changing the understanding of enzyme mechanism and therefore is no longer used as an model example - 35what is the importance of water molecule in protein binding interactions- how has this been shown by crystallography- Water molecules are highly important In recognition and aspartame discrimination by the asparaginyl-tRNA synthetase
- Active site complexed with asparagine where there is a water molecule that makes hydrogen bonds with the carboxylate carbonyl group and the amino group of the asparagine
- Need to distinguish between asparagine and aspartate to ensure the correct amino acid is incorporated and this is where the water helps
- If the carboxylate was present in that position water molecules would not be able to form the 3 hydrogen bonds that are required- instead only 2 would be formed
- Allows fro discrimination between the asparagine and the aspartate - 36give an example of protein protein interactions that has been gained via crystallography-can also be studied using CryoEM
- The GroEL-GroES complex is a large complex that is composed of 2 parts
- The structure of the complex enabled understanding of how it aided the protein folding —> due to the protein dynamics
- Large movements were observed in the complex structure with nucleotides bounds compared to the GroEL alone - 37what is the function of the GroEL/ES as determined by crystallograpic evidenceIn the open form (unbound to the GroES) the protein has 3 domains and is compact
- When in the open form it is able to bind proteins that are misfolded or unfolded
- Upon the binding of proteins the GroES cap is combined and binds ATP as an energy source
- Conformational change within the protein and exposing hydrophilic regions that is then able to help protien folding before it is ejected from the complex - 38how can crystallographic data be used to determine the dynamics of a protein despite being a static methodable to solve the structures of different conformations/states allows for an insight in the protein dynamics
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- Protein structure and function Karteikarten am Protein crystallisation 3, erstellt von Samantha bruce am 06/12/2019.
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- Samantha bruce
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