Lecture 7 Diffraction Data

Question 1

What x ray sources are used in crystallography

Answer 1

X ray generators that have X ray tubes or rotating anodes

Synchrotrons:
- shared factilites that you book in advance to use

Question 2

Explain X ray generators

Why do they have a beryllium window

Answer 2

X ray tube:
- electrons in the tube hit the stationary target anode coated with copper
- this knock electron from the target anode
- x rays come from those electrons decaying and losing energy at characteristic wavelengths
- more energy released is Kbeta which is shorter wavelength

Rotating anode:
- same but anode rotates

Be window:
- encloses the system to create a vaccum
- toxic beryl used because it has the least electrons , this makes it so it interacts the least with the x rays

Question 3

Why do we use nickle to absorb the radiation from x rays

Answer 3

Nickle absorbs more of the kbeta shorter wavelength than the kalpha of the copper radiation

This is used to remove the radiation of kbeta and keep the more intense kalpha radiation/ x rays for our experiment

Don’t want both ray types because you would see multiple of the same diffraction dot but not superposed because the diff wavelength give diff theta

Question 4

Describe the synchrotron in depth

Answer 4

The electrons are bent around the synchrotron and radiation comes off tangentially to the ring

This is where the x rays come from

Question 5

What did they used to use for detectors

What do they use now

Answer 5

Used to use film, but hard because is has to be developed

Now 4 types:
- solid state
- CCD
- image plate
- multi wire detectors

Question 6

Describe the lab setup of the crystallography experiment

Answer 6

• X-rays hit crystal then beam stop
• beam stop made of lead because it has more electrons which stops the X ray
• diffracted rays collected on detector
• stream of N2 to collect data at low temp so reduced crystal decay
• need to rotate the crystal via the goniometer to collect diff data (get more points in ewalds sphere)
• moved by phi (rotate) omega and pi (move up at an angle)

Question 7

What are the types of crystal mounts

Describe them

Answer 7

Capillary mount:
- mounting the crystal within a capillary that is transparent to the x rays and keeps the crystal isolated from the atmosphere

Process:
- have the formed crystal in motherliquor on a cover slip
- capillary filled with solution like the mother liquor so the surface tension is on top
- the density of the crystal is higher then the mother liquor
- when capillary touches crystal, crystal falls to bottom of capillary
- suck out excess liquid from the capillary and break off the top and seal

Cryoloop/cryomount
- this is specially for data collection at low temperatures which makes it so that the solution in the crystal is kept from freezing
- this prevent ice diffraction interference and prevents disruption of the crystal lattice (which could lower protien diffraction and resolution)
- so the crystal is transferred to cryo solution that won’t freeze and is then vitrified

Process:
- crystal on coverslip and loop around same size as crystal scoops up crystal
- need to be fast so small volume of solutions don’t dry out
- cryoprotectant would be in the loop and want the solution around same thickness as the crystal
- after data collection the vitrified solution turns yellow

Question 8

Explain how the detector is positioned in the diffraction experiment

Answer 8

The detector is at an angle of 2 theta from the beam because the crystal diffracts the ray at an angle of 2theta

Also want the detector close enough because if too far the diffraction spots are further apart and some points could be missed

Question 9

What is the very first data they collect at the start of the diffraction experiment

Why

Answer 9

Collect:
- two images of crystal in two positions 90 degrees from each other
- Then they rotate the crystal by 1 or 1/2 degree during the collection of each image

Why:
- These two images are used for auto indexing where a computer will look at the reflections on the images and use that to calculate the unit cells and crystal orientation

Question 10

Explain the autoindexing results

How do they know which result to pick

What assumption are we making

Answer 10

The program gives different options for crystals and their unit cells dimensions and space groups

Also give a penalty based on if the structure they predicted obeys the law of what is should be:
- ex. They predicted orthrombic, meaning alpha beta gamma should be 90 deg, but they aren’t so there’s a penalty for that option

They pick the result that is right before a high jump in penalty:
- assuming the highest symmetry that seems reasonable

Assumptions:
- haven’t actually measured the intensity so don’t actually have the symmetry.
- So we’re assuming that the symmetry/space groups that it says is what we have, but actually the unit cell could just look to have that symmetry and not actually have it
- these unit cell dimensions and orientation of the crystal get refined using more data (we don’t stick with the values auto indexing gives)

Question 11

How is the volume of the unit cell calculated

How can the number of possible reflection be calculated

What happens to diffracted at the edge of the limiting sphere

Answer 11

If it’s orthogonal:
- V= abc (unit cell edges)

Number of measurable reflections (N):
- based on ewalds sphere which has radius 1/lambda
- the limiting sphere (ewalds sphere can’t go past this) is 2/lambda
- volume of the limiting sphere is V= 4/3pi r^3
- N= 33.5V / lambda^3

Diffraction:
- the crystals don’t diffract at the edge of the limiting sphere
- so you decrease the wavelength to collect more reflections

Question 12

What is the solvent content of protien crystals

What is Matthew’s coefficient

What do you need to use Vm

Answer 12

43%

Matthew’s coefficient :
- volume in the crystal per unit of protein molecular weight
- can use the expected value of Vm to guess how many molecules of protien are in an asymmetric unit of crystals
- mean Vm: Vm= 2.61A^3/Da

Need:
- the volume of the unit cell
- how many asymmetric units/symmetry equivalents (based on the space groups)
- size of the protien
- use this info to see how many protien molecules are in the asymmetric unit cells

Question 13

What happens after the unit cell and symmetry are determined

How is the data processed

What are partial measurements

Answer 13

Use computers to decide the best way to collect the data:
- want to collect the unique reflections as quickly as possible
- one data set is 90 images with 1 degrees diff per image

Processed:
- Now youve measured intensities of the reflections and then scale those intensities together
- scale because if rotate the crystal, the intensity would increase way higher if all of it is in the path of the beam, so scale
- Also avg the measurements that have been made more the once

Partial measurements:
- each image taken gives partial fragments of the diffraction peak
- so keep the partial measurements and sum them up
- if crystal is less mosaicity, peak is sharper because the units cell in the crystal are diffracting more together (less fluid)
- so the mosaicity tells the software how many images it needs to sum to get the entire peak aand get the best estimate of the partial reflections