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3006 > 1 - X-Ray Crystallography > Flashcards

1 - X-Ray Crystallography Flashcards

1
Q

What are the four main steps in X-ray crystallography?

A

Growing and mounting the crystal
collecting primary diffraction data
collect data that allow for phase estimation
refine data to produce electron density map and trace chains

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2
Q

What form of crystallography can be used to capture dynamic structures?

A

Time Resolved Crystallography

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3
Q

How does Time Resolved Crystallography work?

A

After mounting the crystal, the enzymatic reaction is activated and diffraction results collected at different time intervals.
Alternately, reactions can be begun and quenched at different times by flash freezing.

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4
Q

What methods can be used to activate a reaction in time resolved crystallography?

A

Photolysis of something (pump probe method) or by addition of substrate or removal of inhibitor (diffusion trapping method).

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5
Q

What are the typical components of crystal growing mixtures?

A 
5-45% polyethylene glycol (PEG)
Ammonium sulphate (reduces solubility, improves order)
NaCl
Mg++
Zn++
alcohols
detergents
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6
Q

What are the two main methods of growing crystals?

A

Hanging drop and Sitting drop

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7
Q

How are mixtures screened for crystal growth?

A

crystal growing robots used to set up large numbers of crystal growth tabs to identify the best solution and method.

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8
Q

What proportion of protein crystal is solvent?

A

Typically 50%, mostly found in channels between unit cells

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9
Q

What are the physical properties of crystals and what does this allow for?

A

Soft and delicate, sensitive to environmental change.

Allows for saturation of crystals with solutions.

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10
Q

What solutions might a crystal be saturated with?

A

metal solutions
ligands
substrates
inhibitors

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11
Q

What is the definition of a unit cell?

A

the smallest parallelepiped that when repeated in three dimensions produces the crystal lattice.

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12
Q

Why can unit cells not contain symmetrical planes?

A

the amino acids all have the same chirality and so cannot mirror one another

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13
Q

What can unit cells be divided into?

A

asymmetrical units

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14
Q

What is the definition of an asymmetrical unit?

A

the smallest unit that when repeated enough times produces the unit cell.

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15
Q

What are space groups?

A

the different ways in which proteins can crystallise in order to form different asymmetrical units and hence unit cells.

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16
Q

How many possible space groups are there?

A

65

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17
Q

What are the three kinds of x-ray source?

A

Rotating anode
synchrotrons
Free electron Lasers

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18
Q

How does a rotating anode source work?

A

a copper anode is heated until electrons ‘boil off’. These are accelerated by an electric field so that when they collide with a metal plate they emit bremsstrahlung (braking radiation) of x-ray wavelength. This must be collimated and filtered.

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19
Q

What advantages are there to using rotating anode sources?

A

It is relatively cheap and can be done in-house. The radiation in low enough in intensity not to damage the sample.

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20
Q

What disadvantages are there to using rotating anode sources?

A

Non-selectable wavelength, cannot be used for SAD or MAD. Generally lower quality data due to the lower intensity?

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21
Q

What limits the intensity of rotating anode sources?

A

The cooling of the rotating anode.

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22
Q

How is synchrotron radiation produced?

A

The ‘Dees’ are in fact many-sided polygons. magnets are aligned at each edge to change the direction of the electrons. When an electron changes direction is emits high energy radiation.

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23
Q

Describe the properties of synchrotron radiation.

A

Each junction between two sides produces a different ‘beamline’. These can be monochromatised and polarised, and each is often fixed at a specific setting. The radiation is of incredibly high intensity.

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24
Q

What advantages are there to using synchrotron sources?

A

High intensity and polarisability/monochromatic. Variable wavelength, allows for MAD studying.

Most commonly used source for crystallography.

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25
Q

What disadvantages are there to using synchrotron sources?

A

High intensity degrades the crystal through thermal damage.

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26
Q

What is used to make crystals more stable for data collection?

A

Cryo-crystallography, in which the crystal is flash frozen using using liquid nitrogen or propane, counteracting the thermal input from the beam.

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27
Q

What is the added advantage of cryo-crystallography?

A

Cold crystals have reduced disorder.

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28
Q

What is crystal disorder?

A

This is a negative property of the final image that is dependent on the slight movement in the chains due to thermal motion and low energy difference stereoisomers (eg rotamers) within the crystal.

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29
Q

What advantages are there to using Free Electron Laser sources?

A

Intensity even higher than synchrotrons, monochromatic and polarisable. Can fire in femtosecond bursts. Some structures produced have been markedly different to synchrotron sourced structures.

30
Q

What disadvantages are there to using Free Electron Laser sources?

A

Degrades the crystal almost instantly, so many crystals may be needed to scan all possible orientations.

31
Q

What happens when the X-rays pass through the protein crystal?

A

Most do not interact, some undergo Thomson elastic scattering due to interaction with electrons.

32
Q

Why does scattering occur with X-rays in protein crystals?

A

Because X-rays have a wavelength of 1A, 0.1nm. This is the approximate length of a covalent bond.

33
Q

Briefly describe Bragg’s Law

A

This law describes the angles and resulting relative phases of the rays depending on the interplanar distance between the electron shells which are scattering them.

34
Q

Why is Bragg’s Law important in crystallography?

A

understanding how the waves are scattered is necessary for interpretation of the data.

The resulting intensity is more dependent on angle of incidence/interplanar distance for shorter wavelengths. using a fixed wavelength therefore necessitates rotating the crystal in order to obtain the right angle of incidence on all areas.

35
Q

What equation defines Bragg’s Law?

A

2d sin0 = nL

d = interplanar distance
0 (theta) = angle of reflection
nL (lambda) = number of wavelengths different

36
Q

What detectors are used in x-ray crystallography?

A

X-ray sensitive film, MAR image plate detector or, far more commonly, an ADSC Charge Coupled Device Image Sensor

37
Q

Describe detection of X-rays by an ADSC CCD image sensor

A

X-rays strike an imaging phosphor plate that converts the signal to an electronic emmision. these are directed through an optical taper and then strike the CCD array.

38
Q

Describe the CCD array structure and properties.

A

four 1in^2 plates in a 2 by 2 array, each with a 1150x1150 pixel resolution.
Incredibly high readout time and resolution.

39
Q

What form does the diffraction pattern take?

A

Concentric rings of dots of different intensities. The number of electrons that impact at each point is used to reconstruct the structure.

40
Q

What is the dynamic range of a sensor?

A

sensors may be sensitive to the difference in intensity only between certain max and min limits, below or above which they cannot tell the difference between more or fewer impacts.

41
Q

What is a fourier transform?

A

A mathematical operation that is used to interpret, separate and identify the source of each of the various signals that might be combined into a convoluted one.

Eg sorting a composite signal into its interfering sine-waves.

42
Q

What two things about the waves are necessary to use the fourier transform to determine the structure, and which has a greater effect on the resulting structure?

A

The amplitude (derived from the diffraction spot intensities)

The phase of the waves. This is far more important for determining the structure.

43
Q

Into what two classes do solutions to the phase problem fall?

A

Isomorphous Replacement and Molecular Replacement

44
Q

What are the principles of isomorphous replacement?

A

Perturbing the crystal structure and comparing the slightly different one to the original. The unit cell size and general protein shapes/angles must be maintained.

45
Q

What are the two types of isomorphous replacement used?

A

MIR - multiple isomorphous replacement

MAD - Multiple-wavelength Anomalous Dispersion

46
Q

What is MIR?

A

Several crystals all perturbed (hence MIR not SIR) by saturation with different heavy metal solutions.
Must be done with different metals to perturb different parts of structure, else areas with no perturbation would be reduce phase solving accuracy.

47
Q

What are the dis/advantages of MIR?

A

Adv. - Can be done in house with rotating anode sources as does not require multiple wavelengths

Dis. - Areas of non-isomorphism can limit resolution. Largely obsolete now.

48
Q

What is MAD?

A

Introduction of anomalous molecules, moieties or properties into a protein to disturb the structure.

Often in the form of Fe-S centres, zinc ions or seleniomethionine, though the latter can cause too much disruption.

49
Q

What are the dis/advantages of MAD?

A

Adv. - High resolution due to excellent phase solving.

Dis. - Must be done in a synchrotron facility ddue to needing multiple wavelengths.

50
Q

What is Molecular Replacement?

A

This relies on using a homologous or structurally similar protein for which the structure is known to make assumptions that allow for phase estimation. At least 40% sequence homology is needed.

51
Q

What is the main drawback with using molecular replacement?

A

Model bias. The influence of the phase of the predicted structure is so overwhelming that if the template is not as similar as predicted it can lead to a detailed but inaccurate structure.

52
Q

For each atom, what information will be contained within the full table of results?

A 
The atom number
elemental identity
residue/residue number
spatial co-ordiantes (x, y and z)
Occupancy
B-factor
53
Q

What is the occupancy of a spatial co-ordinate?

A

The number of atoms that may be filling that space. This may be greater than one due to disorder (i.e. overlapping stereoisomer conformations).

54
Q

What is the B-factor of an atom?

A

a measure of the random thermal motion of the atom, how far it vibrated around its specified position.

55
Q

What atoms are likely to have high B-factors?

A

atoms at the C- or N-terminus

atoms interacting with water molecules

56
Q

What is the accuracy of the B-value dependent on?

A

The occupancy value being correct.

57
Q

What are the three most common types of structure maps?

A

Fo-Fc
2Fo-Fc
Omit maps

58
Q

What is an Fo-Fc map?

A

a type of map that determines the positive and negative electron density using the amplitude and shifts the atoms in the relevant way.

59
Q

What is the purpose of using an Fo-Fc map?

A

To reduce the model bias. Useful for MR experiments.

60
Q

What is a 2Fo-Fc map?

A

Like a Fo-Fc map but does not reduce the model influence as severely, the map is created only by charting positive electron density, with negative density only used when the model is significantly different.

61
Q

What is an omit map?

A

Omit maps are used to remove atoms that are being incorrectly modelled from the equation so that they do not contribute to the overall phase solution, allowing the rest of the model to be more accurate. Useful for MR.

62
Q

What is Procheck?

A

An example of software that checks to see if the predicted structure complies with what is already known about the protein and biophysical possibility.

63
Q

What does software like procheck check?

A

Whether or not the torsion angles are typical.
Whether the data agree with the ramachandran plot
Whether bond lengths, angles and van-der-waals contacts are consistent with databases.

64
Q

What does the R-factor measure?

A

The level of discrepancy between the refined model and the initial diffraction data.

65
Q

What derivative factors can be found from the R-factor?

A

R-free and R-sym

66
Q

How is R-free determined?

A

Similar to the R-factor but uses a random diffraction data sample that is not used in the fourier transform.

67
Q

How does R-free compare to the R-factor?

A

R-free is always higher than the R-factor.

The discrepancy between the two is larger if there is more ‘overmodelling’ of the data.

68
Q

What is R-sym?

A

A way of measuring the accuracy of the structure by looking at how well the diffraction data from opposite (180) sides of the crystal agree with one another.

69
Q

What values of R-sym would be deemed too high for the structure to be valid?

A

over 10%

70
Q

What values of R-free would be deemed too high for the structure to be valid?

A

over 20%

3006 (6 decks)

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