Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BS3070 > X-ray crystallography > Flashcards

X-ray crystallography Flashcards

1
Q

What percentage of structures in the pdb have been solved by X-ray?

A

over 90%

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is X-ray diffraction?

A

An X-ray wave hits the body creating scattering, these scattered waves can be converted into an image

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

When is the resolution limit reached?

A

When two point like objects can be imaged as 2 distinct images

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the microscopes resolution?

A

The shortest distance between two points at which they can still be separated and observed as 2 points

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the rayleigh criterion?

A

The criteria for the minimal resolvable detail in which the first darkening of the airy disk is coincident with the peak of a nearby airy disk

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How does numerical aperture affect resolution?

A

High numerical aperture gives high resolution

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Why use crystals as opposed to an individual protein?

A

X-ray scattering is correlated to electron density therefore the scattering from a single molecule is very weak, thus using a repeated ordered array of the molecule gives a concentrated scattering

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the reciprocal lattice?

A

The diffraction pattern of the lattice

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How can the reciprocal lattice be used?

A

From the size of the spots the size of the repeating unit can be determined, so the smaller the spot the larger the repeating unit. From this the intensity of each spot can be measured and assigned an individual index, along with the phase we can then relate the sum of all the reflections to the electron density using the fourier transformation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the fourier transform?

A

Dissects the wave into components that make it, splitting the wave into circular paths but we need the phase which is lost during measurement

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How do we interpret and refine structures?

A

Use our prior knowledge:

  • We know that proteins are chains therefore they must join up
  • We know the approximate structure of a peptide, therefore the bones of the polypeptide can be calculated
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the resolution at which each atom can be placed to within 0.1A?

A

1.5A

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the resolution at which we lose any structural information?

A

4A

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is a temperature (B) factor?

A

Shows the movement of the particle due to temperature, the larger the B factor, the less defined the position is

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is the average B factor?

A

15-30

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What does a Ramachandran plot show?

A

The allowed and disallowed angles for a bond

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is R-sym?

A

Crystals often have symmetry, this allows several observations to be collection from one sample

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What is I/sigma?

A

The relationship between the strength of the measured data and an estimate of the errors will give an indication of reliability

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How are crystals developed?

A

Through slow removal of the protein from solution

Two main methods: Vapour diffusion and Dialysis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Outline crystal growth through dialysis

A

Large molecules can’t diffuse through the small pores in the semi-permeable membrane, therefore the [protein] remains constant on one side. Then the pH/buffer is changed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is the main disadvantage of growing crystals through dialysis?

A

Uses alot of protein about 10mg/ml

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Outline the Vapour diffusion process of growing crystals

A

Reservoir solution contains buffer and precipitant.
Vapour diffuses off the drop of protein to the reservoir solution over time until the concentration of the precipitant is equal in both

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What is the main advantage of vapour diffusion?

A

It requires very little protein meaning that a variety of conditions can be screened

24
Q

What is the ideal zone for crystal growth?

A

In the precipitation zone, near the border to the nucleation zone

25
Q

Outline the traditional method of putting crystals in the beam

A

Draw crystal into a thin walled capillary, the crystal sticks to the side through surface tension, then seal the capillary with wax and the relevant buffer.
Mount with a goniometer which allows fine adjustments in the beam

26
Q

What are the main disadvantages of the traditional method of positioning the crystal?

A

Extra scattering due to the capillary and liquid

Difficulty in manipulating the sample

27
Q

What is the modern approach for positioning the crystal?

A

Pick the crystal up with a thin fibre loop (10µm) rayer or nylon and cool it rapidly to <100K

28
Q

What is the main disadvantage of the modern approach for positioning the crystal?

A

Conditions need to be found that will not form ice crystals

29
Q

What are the advantages of the modern approach for positioning the crystal?

A

Very little extra material is being put in the X-ray beam
Very little manipulation
Crystals last and can be stored

30
Q

How is the data collected?

A

The crystal is mounted carefully into the beam and a stream of nitrogen maintaining the temperature at about 100K. The crystal is oscillated about a small angle and the resultant diffraction recorded. The intensity of each reflection is then measured and a list of their reflections and their value made.

31
Q

What are the three main methods of X-ray generation?

A

X-ray tubes
Rotating anodes
Synchotrons

32
Q

Outline how X-ray tubes produce X-rays

A

In a sealed tube a current heats a filament causing thermionic electron emission. This is then accelerated by a large potential difference of about 40kV, to strike a target anode releasing blackbody radiation, 1% of which is X-ray

33
Q

Outline how a rotating anode produces X-rays

A

In a sealed tube a current heats a filament causing thermionic electron emission. This is then accelerated by a large potential difference of about 40kV, to strike a target a rotating anode releasing blackbody radiation, 1% of which is X-ray

34
Q

Outline how a synchotron generates X-rays

A

Electrons are produced by thermionic emission and accelerated by electric fields in a linear accelerator until they reach 99.99% the speed of light, when they are injected into a ring for storage where they orbit, through deflection by bending magnets. Radiation is then tapped at these bending magnets, when required

35
Q

What are the ways of creating monochromatic X-ray beams?

A

Filters
Single crystal monochromators
Double crystal monochromators

36
Q

Describe X-ray filters

A

Use a foil of an element with an atomic number of 1 less than your filament

37
Q

Describe single crystal monochromators

A

Use crystals made out of graphite of silicon to reflect wavelengths that obey Bragg’s law for the particular spacings of the monochromator

38
Q

Describe a double crystal monochromator

A

2 parallel crystals that rotate to amplify a specific wavelength without altering the direction of the beam

39
Q

What surface can bend X-rays at low angles?

A

Nickel coated glass

40
Q

What are the various forms of detection?

A

Image Plates
Multi-wire proportional counters
CCDs
Pixel Detectors

41
Q

What are image plates?

A

Stage phosphate, when an X-ray strikes it converts Eu2+ –> Eu3+.

Red light from a HeNe laser causes the release of a blue photon that can be picked, measured with a photo-multiplier and then digitized

42
Q

What are multi-wire proportional counters?

A

Xenon chambers with a trace of methane under pressure with a high voltage applied. Photons hit a Xenon atom ionizing it. The Xe ion is then accelerated towards the cathode, along the way it collides with more Xe atoms causing more ions to form. Under the right conditions the response should be proportional to the number of photons

43
Q

What is a CCD?

A

A solid state device that separates the charge caused by an incident photon on the silicon substrate

44
Q

What is the phase problem?

A

The phase is lost during measurement however it is required for the fourier transform to get meaningful data.

45
Q

How is the phase problem fixed?

A

Multiple isomorphous replacement

Multiple anomalous dispersion

46
Q

What is a patterson map?

A

A map of vectors between the atoms , created by applying the fourier transform to intensities alone

47
Q

Describe multiple isomorphous replacement

A

The protein sample is soaked in a solution containing heavy metal ions, which bind to the protein at certain positions. Then compare different diffraction patterns with and without the ions, thus allowing us to work out the positions of the ions

48
Q

Describe multiple anomalous dispersion

A

Heavy metal ions are incorporated by soaking in metal ions or by using selanomethionine. Then choose a wavelength that the metal ions absorb due to resonance therefore their location can be determined by the ‘holes’ they leave in the image

49
Q

How is multiple isomorphous replacement data converted to the phase?

A

The Harker construction which finds two possible phases from each derivative. Uses a circle to compare and only one phase choice will be constant for all derivations

50
Q

How is multiple anomalous dispersion data converted to phase?

A

Anomalous dispersion
If we hit the subject at the right energy the photon will resonate causing a small change in the amplitude and phase of the scattered wave. This can then be put through harker constraints

51
Q

What are the point group symmetry operations?

A

Rotation Axes
Inversion Axes
Mirror Plane

52
Q

What are the different forms of structure validation?

A 
The Wilson plot
Real space R value
Real space correlation coefficient
Z scores
Temperature factors
Data anisotropy
Procheck
53
Q

What is the Wilson plot B?

A

the value shows the average for all atoms based on how the intensity of the diffraction data drops off with resolution

54
Q

What is the real space R value?

A

The difference between the calculated density and the observed density

55
Q

What is the real space correlation coefficient?

A

Similar to the R value except that the linear correlation coefficient between the density arrays is calculated

56
Q

What are Z scores?

A

Show how real space R scores compare to resolution

57
Q

What is data anisotropy?

A

The analysis of the data from different directions

BS3070 (4 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions