• 38:20
• Fourier transform can interconvert structure to spots and vice versa, but only if we have the phase (as we must know Fhkl where hkl are the coordinates of the spot and F involves all the other mathematical information such as the phase and intensity). We know the wavelength, amplitude but cannot determine the phase. So we make a number of educated guesses of the phases and then refine them.
§ First solution for phase problem is the
aforementioned multiple isomorphous replacement (MIR).
Soak in heavy atoms introduced as salts to the crystals.
Leaves the diffraction pattern the same, it is an
isomorphous derivative (structure unchanged), but changes amplitude of certain spots. Hence, there are simple
structures in the cell that scatter x-‐rays strongly. We can predict how these strong scatterers will affect the
amplitudes. By doing this a couple of times with a couple different atoms we can back calculate the phase of each spot.
§ Second solutions. More recently we use molecular replacement. If there has been a related structure solved, if it shares > 30% sequence homology (we hope that there is structural homology too), we can orient it within the unit cell and then change the orientation/position and back calculate the intensities until intensity looks like what we observed in our experiments. When we get good overlap we can be pretty sure we have something that looks like our
What information does Fourier transformation require and how is it obtained?
What is the phase problem and two solutions?
experimental crystal structure, we can then calculate phase from that, and use this to create a new electron density map, and if this has features that are interpretable/that we can build into, then we can start solving the structure.
§ Step 4: refinement and construction of the structural model: use specialised computer programs to refine the electron density map and optimise the position of atoms in this map. Also add water molecules as these contribute to phase information. Can monitor the refinement of this with Rfree which is a measure of how well the model agrees with the diffraction pattern
§ 46:19 Coot (model building) o Last step is always to submit to PDB
o Electron density modeling: Sometimes the mutations you make in the enzyme to study it can allow for different things to bind and produce strange electron density maps.
Lecture 13
o How do proteins transmit signals?
§ There is usually a signal outside the cell, such as a hormone, that must be transmitted into the cell. Cells have evolved receptors (transmembrane proteins). Binding of the outside signals causes a conformational change in the protein that affects its internal domain.
§ We will be looking at SH2, 3 and PH domains. These are all between 60 and 150 residues in size, and can all bind different proteins. PH is more so a lipid binding domain.
These three are common in intracellular proteins.
§ Where as Fn3 and Ig like domains are seen more in receptor proteins.
§ We will also look at protein kinases. CDK is two domains come together to make a single structure.
§ They are regulated by phosphorylation, however there is certainly more detail than that.
§ Receptor mediated signalling will also be covered, where hormones bind, receptors dimerise, autophosphorylate and we will look into deeper detail than that also.
§ We will look at receptor kinases and non-‐receptor kinases.
§ Small angle scattering will be the technique section of Mulhern's lectures, and shares a lot of similarities with X-‐
ray crystallography in equipment and set up, just patterns are different. SAXS requires a greater distance due to small angles of diffraction, as the name suggests. Where as X-‐ray crystallography does not require so much distance.
How can you monitor the refinement of the structure?
What do you do once you have performed the four steps of solving a crystal structure?
