X Ray 2 Flashcards
What are the first steps in the x ray crystallography experiment
Produce and purify the protein
Grow the protein crystal
Expose that crystal to x rays and measure the diffraction pattern
For the first steps in x ray crystallography why cant we take the diffraction patterns and reconstruct the model from that like we do with a microscope
We don’t have the correct lenses for that
What are the next steps in x ray crystallography
After getting the diffraction pattern you Collect data about
The phases
That gives you an electron density map
From that you can build an initial model
Then you refine that model
What two characteristics does a wave have
Amplitude and phase
How do we get the amplitude of a wave from x ray crystallography
It’s comes from the intensity of the x rays
When we get those black dots and add up all the photons that are coming from that phase we get the intensity which gives amplitude
What is the phase problem
You can’t measure the phase of the x rays
What are the two major difficulties in x ray crystallography
Growing the crystals
The phase problem
What is a Fourier transform
An operation that gets us from one to the other
Can take us backward and forward
Ex. Going from the amplitude and phases from the diffraction pattern to the electron density map
Or getting the amplitude and phases from the diffraction pattern from the electron density map
What concepts are important to know for a Fourier transform
Sampling
Importance of phases
Why the estimates of phases are useful
Resolution
In a Fourier diagram (the rainbow sphere) what is the amplitude represented by
What is the phase represented by
Amplitude is represented by the colour saturation and brightness
Phase is represented by the hue
What does phase angles does a hue of red green and blue correspond with
0 degrees
120
240
In the forums rainbow sphere what is the reference point that weed use to measure our phases from
The real axis (x axis) which is zero degrees
If you have a real space diagram of a single amino acid what does it look like
What does the Fourier transform of it look like
Ex. For histidine in real space we’d see the electron density centered at each atom
In the Fourier transform you get an array of colours which correspond to the phase
And the color fades out
Ex. In the middle is very red so the phase is zero
If you have a real space diagram of a amino acid lattice (due to being crystallized ) what does it look like
What does the Fourier transform of it look like
So now it’s a repeating unit (an array of molecules) in real space
In transform you see three things:
There is sampling
There are discrete points because of the repeating unit
The longer repeat in the transform (horizontal) is opposite of what it was in real space (vertical) meaning there something in that repeat that we see in the Fourier transform
In Fourier transform what is more important in getting the real space image
Give an example why
The phases are more important than the amplitudes
If we mix the amplified from a duck with the phases from a cat, we see the cat image
Why is the fact that phases give more info for the image bad
because in x ray crystallography we measure the intensities to get amplitude
We don’t actually measure phases because we don’t see the coulour in the Fourier transform
Do the phases in Fourier have to be perfect to see that image in real space
Why
No
If we just have the amplitudes from a cat and we have the known phase of a Manx
When we mix we get the Manx cat but with a faint tail
So even though most info to get the image is coming from the phases, if you have phases that are close, the amplitude can help distinguish that part the we were missing the phases for
Give a biochemical example of how we use the phase of a different but similar sample to get the image of our sample
If we have an inhibitor bound to an enzyme and they crystallize the same way
The tail would be the inhibitor and the original protein that we already know the Sri cure for is the Manx cat
Once you have estimates of the phases what can you do but what is the problem
You can make electron density maps
But they won’t be perfect since you estimated the phases
If we use low resolution data in a Fourier transform what happens
This means you truncated the Fourier transform
So You get low resolution image in real soace
Higher resolution means
Smaller numbers
What is a fitting model
When you build the amino acids into the electron density
Then you can get atomic coordinates (xyz)
Ex. A phenylalanine is moved into the electron density because it look like it should be there
What is crystallographic refinement
Improving the model so that we get a better match between the observed intensities (from our data) and those calculated from the model
Minimizing the difference between what we observed vs what’s actually calculated from the model
In crystallographic refinements what are the parameters we can refine
The atomic coordinates for each atom (x,y,z) (the atom can be moved around to get a better match to the observed data
Giving an extra parameter to each of our atoms: the atomic motion (temperature factor B) which describes different positions of the atoms in space
Occupancy (if the atom is there) which is usually 1 (if not there it’s 0)
How many parameters for each atom are we refining in crystallographic refinement
4 parameters
Slide 21
Idk
What is the problem with the amount of data you have and the refinement
You generally don’t have enough data points for the refinement to be stable
You’d need other information
What do we use to judge the quality of the crystals in x ray crystallography
The diffraction pattern of the crystals
If the resolution of our data from the diffraction pattern was not good enough
we’d only see a few diffraction spots in the middle of the pattern and need to grow better crystals
Why do we know a lot about what isoleucine would look like
Because it’s in small molecule crystal structure and we can get small molecule crystal structures at high resolution
What can we use the bond length bond angle and torsion angle for in. Refinement
What else can be used for refinement
Can use that info as restraints by weighting each term
Can add in the van der walls repulsion term to keep atoms apart since they could go into the same place in the electron density
Can do molecular dynamics refinement: allowing the atom to move to different positions to get a better match to the data
Include crystallographic data you collected
When do we stop refining our model
What does this mean
When there are no more improvements to be made
It’s an iterative process
How does an NMR person know when their model is done to submit it to the protein data bank
Keep collecting data until they get a specific root mean squared deviation
Or until they solve their biological question
