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BS3070 > X-ray crystallography > Flashcards

X-ray crystallography Flashcards

(57 cards)

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1
Q

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

A

over 90%

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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

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3
Q

When is the resolution limit reached?

A

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

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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

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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

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6
Q

How does numerical aperture affect resolution?

A

High numerical aperture gives high resolution

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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

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8
Q

What is the reciprocal lattice?

A

The diffraction pattern of the lattice

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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

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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

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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
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12
Q

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

A

1.5A

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13
Q

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

A

4A

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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

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15
Q

What is the average B factor?

A

15-30

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16
Q

What does a Ramachandran plot show?

A

The allowed and disallowed angles for a bond

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17
Q

What is R-sym?

A

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

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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

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19
Q

How are crystals developed?

A

Through slow removal of the protein from solution

Two main methods: Vapour diffusion and Dialysis

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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.

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21
Q

What is the main disadvantage of growing crystals through dialysis?

A

Uses alot of protein about 10mg/ml

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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

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23
Q

What is the main advantage of vapour diffusion?

Study These Flashcards
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?

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A

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

25
Outline the traditional method of putting crystals in the beam
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
What are the main disadvantages of the traditional method of positioning the crystal?
Extra scattering due to the capillary and liquid | Difficulty in manipulating the sample
27
What is the modern approach for positioning the crystal?
Pick the crystal up with a thin fibre loop (10µm) rayer or nylon and cool it rapidly to <100K
28
What is the main disadvantage of the modern approach for positioning the crystal?
Conditions need to be found that will not form ice crystals
29
What are the advantages of the modern approach for positioning the crystal?
Very little extra material is being put in the X-ray beam Very little manipulation Crystals last and can be stored
30
How is the data collected?
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
What are the three main methods of X-ray generation?
X-ray tubes Rotating anodes Synchotrons
32
Outline how X-ray tubes produce X-rays
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
Outline how a rotating anode produces X-rays
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
Outline how a synchotron generates X-rays
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
What are the ways of creating monochromatic X-ray beams?
Filters Single crystal monochromators Double crystal monochromators
36
Describe X-ray filters
Use a foil of an element with an atomic number of 1 less than your filament
37
Describe single crystal monochromators
Use crystals made out of graphite of silicon to reflect wavelengths that obey Bragg's law for the particular spacings of the monochromator
38
Describe a double crystal monochromator
2 parallel crystals that rotate to amplify a specific wavelength without altering the direction of the beam
39
What surface can bend X-rays at low angles?
Nickel coated glass
40
What are the various forms of detection?
Image Plates Multi-wire proportional counters CCDs Pixel Detectors
41
What are image plates?
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
What are multi-wire proportional counters?
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
What is a CCD?
A solid state device that separates the charge caused by an incident photon on the silicon substrate
44
What is the phase problem?
The phase is lost during measurement however it is required for the fourier transform to get meaningful data.
45
How is the phase problem fixed?
Multiple isomorphous replacement | Multiple anomalous dispersion
46
What is a patterson map?
A map of vectors between the atoms , created by applying the fourier transform to intensities alone
47
Describe multiple isomorphous replacement
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
Describe multiple anomalous dispersion
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
How is multiple isomorphous replacement data converted to the phase?
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
How is multiple anomalous dispersion data converted to phase?
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
What are the point group symmetry operations?
Rotation Axes Inversion Axes Mirror Plane
52
What are the different forms of structure validation?
``` The Wilson plot Real space R value Real space correlation coefficient Z scores Temperature factors Data anisotropy Procheck ```
53
What is the Wilson plot B?
the value shows the average for all atoms based on how the intensity of the diffraction data drops off with resolution
54
What is the real space R value?
The difference between the calculated density and the observed density
55
What is the real space correlation coefficient?
Similar to the R value except that the linear correlation coefficient between the density arrays is calculated
56
What are Z scores?
Show how real space R scores compare to resolution
57
What is data anisotropy?
The analysis of the data from different directions

BS3070 (4 decks)

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