X Ray 2

Question 1

What are the first steps in the x ray crystallography experiment

Answer 1

Produce and purify the protein

Grow the protein crystal

Expose that crystal to x rays and measure the diffraction pattern

Question 2

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

Answer 2

We don’t have the correct lenses for that

Question 3

What are the next steps in x ray crystallography

Answer 3

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

Question 4

What two characteristics does a wave have

Answer 4

Amplitude and phase

Question 5

How do we get the amplitude of a wave from x ray crystallography

Answer 5

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

Question 6

What is the phase problem

Answer 6

You can’t measure the phase of the x rays

Question 7

What are the two major difficulties in x ray crystallography

Answer 7

Growing the crystals

The phase problem

Question 8

What is a Fourier transform

Answer 8

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

Question 9

What concepts are important to know for a Fourier transform

Answer 9

Sampling

Importance of phases

Why the estimates of phases are useful

Resolution

Question 10

In a Fourier diagram (the rainbow sphere) what is the amplitude represented by

What is the phase represented by

Answer 10

Amplitude is represented by the colour saturation and brightness

Phase is represented by the hue

Question 11

What does phase angles does a hue of red green and blue correspond with

Answer 11

0 degrees

120

240

Question 12

In the forums rainbow sphere what is the reference point that weed use to measure our phases from

Answer 12

The real axis (x axis) which is zero degrees

Question 13

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

Answer 13

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

Question 14

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

Answer 14

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

Question 15

In Fourier transform what is more important in getting the real space image

Give an example why

Answer 15

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

Question 16

Why is the fact that phases give more info for the image bad

Answer 16

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

Question 17

Do the phases in Fourier have to be perfect to see that image in real space

Why

Answer 17

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

Question 18

Give a biochemical example of how we use the phase of a different but similar sample to get the image of our sample

Answer 18

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

Question 19

Once you have estimates of the phases what can you do but what is the problem

Answer 19

You can make electron density maps

But they won’t be perfect since you estimated the phases

Question 20

If we use low resolution data in a Fourier transform what happens

Answer 20

This means you truncated the Fourier transform

So You get low resolution image in real soace

Question 21

Higher resolution means

Answer 21

Smaller numbers

Question 22

What is a fitting model

Answer 22

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

Question 23

What is crystallographic refinement

Answer 23

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

Question 24

In crystallographic refinements what are the parameters we can refine

Answer 24

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)

Question 25

How many parameters for each atom are we refining in crystallographic refinement

Answer 25

4 parameters

Question 26

Slide 21

Answer 26

Idk

Question 27

What is the problem with the amount of data you have and the refinement

Answer 27

You generally don’t have enough data points for the refinement to be stable

You’d need other information

Question 28

What do we use to judge the quality of the crystals in x ray crystallography

Answer 28

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

Question 29

Why do we know a lot about what isoleucine would look like

Answer 29

Because it’s in small molecule crystal structure and we can get small molecule crystal structures at high resolution

Question 30

What can we use the bond length bond angle and torsion angle for in. Refinement

What else can be used for refinement

Answer 30

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

Question 31

When do we stop refining our model

What does this mean

Answer 31

When there are no more improvements to be made

It’s an iterative process

Question 32

How does an NMR person know when their model is done to submit it to the protein data bank

Answer 32

Keep collecting data until they get a specific root mean squared deviation

Or until they solve their biological question