Stephen Curry - X-Ray Crys. Flashcards
What topics are covered in this 7 part lecture series?
Outline of Lecture series
- Why use X-rays?
- How is structural information encoded in X-ray diffraction patterns
3, Why do we need to use crystals?
- How to optimise proteins for crystallisation
- How to grow protein crystals
- X-ray data collection
- Solving and refining the structure
Why determine the structure of MNV NS6pro?
Why can’t we use a light microscope to see molecules?
But if we can’t use an X-ray microscope, how do we obtain our images?
But what is a crystal?
What determines the shape of molecules?
What is an X-ray? What are it’s constituent components?
What happens when an X-ray collides with an electron?
With a simple 2 electron system - how do they scattered X-ray interact?
What does diffraction refer to?
What physics experiment can electron X-ray diffraction be compared to?
Does the diffraction pattern collected, somewhat resemble a molecule?
Do crystallographers use degrees or Radians?
Why are we talking about circles? Why do crystallographers care about circular motion?
What are the properties of waves? How are they represented graphically and in a equation?
What does constructive interference mean when adding waves?
What does destructive interference mean when adding waves?
How do waves normally interact, constructively or destructively?
In a hypothetical 2 electron system, how would two in-phase incident beams scatter from 2 electrons (same angle - so interaction occurs) but the two electrons aren’t exactly in the same positon?
Outline what is going on in this system?
How could you calculate the path difference in this scenario?
Basically, how much more the top wave had to travel relative to the bottom wave
How can we use the path difference to calculate the phase difference that arises as a result of the path difference?
How can the scattering be defined using vector algebra? What is the main thing to take away from the eqaution?
What equation can be used to calculate the phase difference in this two electron system? What is it dependent on?
How can the general wave equation be modified and applied to to calculate the resultant wave from our 2 electron system?
How can the equation for the resultant wave in 2θ direction be re-written in a simple format?
Hint - Think ‘sum of’
Can we extrapolate our view of a 2 electron system to a 9 electron system, or even bigger? If so, what can do they all have in common?
YEAHHHH BUDDDYYY - we can apply our resultant wave equation to electron scattering in 2θ direction with amplitude 1 for as many electrons as we need!
Example attached - 9 scattered waves interacting
Important takeaway…
The phase shifts produced will be dependent on the position of the electrons and thus the structure!
The resultant wave produced with be reflective of the electrons in the structure
What is the structure function?
Same thing we were looking at before, having a mathematical description of resultant waves
Are electrons in a real multi-electron structure fixed just like we previously examined in our 2 & 9 electron structure?
How does scattering in a real molecule look like?
How can we breakdown the scattering from a molecule to make it simple and mathematically manageable?
Watch out, there is one extra thing we need to consider when working with different electron densities…
How can we change our F(S) wave equation so that represent the total scattering in one direction in our multi-electron model?
Relationship between p(r) and f(s)?
What is a more ‘proper’ way of representing the the molecule f(s) equation?
Why in reality do we care about the inverse fourier transform of f(s)?
Summary of all the shit in lecture 2 - bringing it all together?
How does a real diffraction pattern look like?
Can we apply our mathematical model to single molecule of protein and obtain all our structural information?
How do crystallographers visualize their crystals? How do they break it down?
What the relationship between the angle of incidence & reflection in a mirror?
Does a crystal made up of molecule reflect like a mirror?
Equation to relate the scattered waves from the adjacent layers in a molecule? What does this equation tells us about when constructive interference occurs?
Braggs Law
When do we get destructive interference accroding to Braggs Law?
Why in many cases do constructive waves not produce a resultant wave that is detected?
Distant layers interact –> Braggs Law needs to hold overall
Why do we rotate the crystal when capturing X-ray diffraction ?
Crystalline diffraction - takeaway messages?
Breakdown the following mathematical equations - why do the differences arise between molecule and crystal?
In reality what would the scattering pattern from a single molecule look like?
How does Molecular vs crystalline scattering differ?
What does consequences of Braggs law when it comes to data collection? What happens in the real experiment?
In reality what is the setup for crystal X-ray diffraction?
Does every dot contain information scattering information from every single molcule in the crystal?
BOOOYEAHHH
Every dot that is present on the film contains scattering from every single part of every single molecule, from every unit cell in the crystal
The inverse Fourier transform allows us to deconstruct that to obtain the structure
Summary of Lecture 3 - How structural data is encoded in X-ray diffraction data?
Is growing protein crystals easy?
What is the first step for growing crystals?
Step 1: get lots of purified protein (lots = at least a few milligrams)
What are the two possible sources of protein for crystallization?
What expression system is commonly used by crystallographers? Benefits/disadvantages of this system?
How can we use recombinant DNA technology to make your protein more “crystallisable”?
What happens if you don’t know the structure? How can we modify the protein to make it more crystallizable?
How can we use Bioinformatics tools to help predict protein structure when trying to produce optimal proteins for crystallization?
What other two thigns can be consider to optimize crystallisability?
Look at paper?
But as we all now in Prof Curry’s wise words, Crystallisation, like life, can be fickle. Why was this the case in his paper published on the NS6 protease? What unexpectadely happened during crystallization attempts?
What is the general approach when trying to crystallize your proteins?
Outline the vapour diffusion technique used for crystallization?
What is the basic theory of protein crystallization theory - what are the different states/levels?
According to the crystallisation, what would be the better outcome (series of event) for crystallization?
What drives the proteins to align themselves in the same orientation across the entire crystal?
We don’t have an explanation to this question we just now that it is possible from experimental evidence and we know that we can change the probability of crystallization by using different conditions.
Furthermore, we know that proteins that are rigid have fixed domain are more likely to have a similar conformation in space - means that it is more likely to crystallize correctly.
Using the crystallisation: theory, explain how we can get microcrystals.
Using the crystallisation: theory, explain how we can get no crystals formation at all?
What are some practical issues to consider when trying to crystallize proteins?
Out of the images attached, which one represent good crystallization?
How has technology helped increase the speed at which we can obtain crystals?
What are the properties of a protein crystal?
Why is the presence of a solvent in crystals useful?
When using crystals what is the max theoretical diffraction angle?
How can we rearrange Braggs Law to make d (dmin) the subject? Definition of dmin and its relationship with the angle of diffraction?
How can we visualize/explain this concept of higher diffraction angle/smaller d-spacing yielding higher resolution?
What is one factor that impacts our ability to get high diffraction angle/high resolution data?
Consequence of mosaic blocks not aligning properly in terms of our dmin/resolution?
On the detector where can we find the high resolution data?
How are X-rays made in the Lab and synchrotron?
How do we collect data in practice - what we need to do when collecting data?
How does cryo-cooling prevent radiation damage?
What is the phase problem?
What are the three pieces of information we need to create an electron density map?
What are the two main errors with crysallization?
Now that we have obtaine the following electron density map…
- What do the different contour lines mean? What do the colours represent
- What do we do moving forward?
Why is obtaining high-resolution data important for fitting the atomic model of the protein to the electron density map?
What does manual model building entail?
Starting point
What does the process of model refinement entail?
What is the Observations to parameters ratio - why is it important for building our model?
Relationship between resolution limit and obs/par ratio? Why is it less of a problem than we would expect?
What stereochemical information can we use to increase the Obs/Par ratio?
What is the ultimate goal of model refinement? What value we trying to minimize?
How does refinement occur in practise?
What does having a high B factor mean?
What are the trend in B factor across an entire molecule?
After postional and B factor refinement, what additional refinement step can be included if you have a high resolution structure?
What is Rmodel (Rwork), what is it used for?
After each refinement “macrocycle” what are the things you should check/look out for?
How do you know when you are finished building your model?
What are the two main parts to a table of statistics in a crystallography paper?
Statistics for data collection and model refinement
- Data collection - quality of X-ray data + interesting properties
- Model refinement - statistical refinement of the model
Outline the meaning of each row in the data collection section of the table.
Outline the meaning of each row in the model refinement section of the table.
Main two limits of crystallography?
Purpose/outline of lecture 7?
What is a preprint?
SARS-CoV 2 protease case study - Title and authors?
SARS-CoV 2 protease case study - Purpose of the abstract? What did the authors try and do?
SARS-CoV 2 protease case study - The introduction?
SARS-CoV 2 protease case study - Results (1)?
SARS-CoV 2 protease case study - Results (2)?
SARS-CoV 2 protease case study - Results (3)?
SARS-CoV 2 protease case study - Conclusion?
SARS-CoV 2 protease case study - Materials & Method?
SARS-CoV 2 protease case study - The stats?
What does the future of structural biology hold?
Why does the highest resolution give the weakest spots?
Why is the background of a detection plate not white?
Why do the highest diffraction patterns correspond to the highest resolution?
And
Why is a smaller ‘d’ value correlated with higher resolution?
What happens when we use other ‘n’ values (orders of diffraction)?
What is streak seeding?
Why does the high resolution data in the X-Ray crystallography data table always have higher errors?
*Lower Signal to Noise Ratio
How can you determine if your crystal structure is correct?
