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Protein Structure and Analysis > X-Ray Crystallography > Flashcards

X-Ray Crystallography Flashcards

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1
Q

What is X-ray crystallography?

A

X-ray crystallography is a way of taking ‘photographs’ of a molecule using X-rays.
Electrons from the atoms scatter the X-rays, so we see the electron cloud around the molecule, and we can use this data to build a model of the atom positions and interpret the image.

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2
Q

Why are X-rays of ~ 0.5 to 1.5 Å used?

A

In order to see a detail x meters in extent, you have to use a radiation no more than double that size

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3
Q

Steps in solving protein structure:

A
  1. Purify protein (10mg or more)
  2. Grow a crystal
  3. Collect and process diffraction data
  4. Phase the diffraction data
  5. Calculate and electron density map
  6. Build and refine the structure
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4
Q

What is a good protein sample?

A

Defined buffer
Defined concentration
No aggregation

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5
Q

A crystal acts as an _

A

Amplifier
They arrange molecules in same orientation, summative effect on waves

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6
Q

A crystal unit cell is defined by its cell constants which are…

A

Edges: a, b, c
Angles: α, β, γ

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7
Q

What is point group?

A

Symmetry of a finite object

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8
Q

Combining point group (symmetry of a finite object) and Bravais lattice symmetries generates _ symmetry

A

Space group

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9
Q

What is the space group?

A

Complete description of the symmetry of an (ideal) crystal.
Knowing the space group, and the contents of the asymmetric unit, defines the positions of all atoms in the crystal.

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10
Q

How is the space group described?

A

Described by letter for shape of lattice and number of molecules per rotation (i.e. P2).

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11
Q

When X-rays interact with crystals _ diffraction occurs from planes in the crystal

A

Coherent

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12
Q

Are darker spots more or less reliable?

A

More

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13
Q

The set of planes will give rise to what?

A

A diffraction spot
Containing diffraction from all parts of the protein

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14
Q

What is the asymmetric unit?

A

The unit cell is divided into a number of identical ASUs
ASUs combine through space group-specific symmetry operations to generate the unit cell

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15
Q

In protein crystallography we determine the structure of the _

A

ASU

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16
Q

What does Mathews Coefficient tell us?

A

About the crystal volume per unit of protein

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17
Q

Low vs high solvent crystals

A

Low solvent content crystals tend to be highly ordered and diffract well.
High solvent content crystals tend to be less ordered – fewer crystal contacts – leading to weaker diffraction

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18
Q

Typical crystalising agents

A

Salts
Long chain organic polymers
Organic solvents

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19
Q

Why does high salt precipitate protein?

A

The salt ions order water molecules around them, leaving less unstructured water to solubilize the protein

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20
Q

Why do organic solvents precipitate protein?

A

They effectively dilute water with a less polar, less H- bond capable solvent with lower dielectric etc.

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21
Q

Why do long chain organic polymers precipitate protein?

A

PEG prefers to writhe over a large volume of space
Taking the protein out of solution frees up more space for PEG and is energetically favoured

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22
Q

Other factors that can effect crystallisation

A

Concentration of protein
Changing pH
Temperature
Ligands (conformational locks)

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23
Q

Why does pH effect crystallisation?

A

Because it changes the number of protons, which can effect salt bridges and H-bonds

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24
Q

How to improve size and diffraction?

A

Systematic variation of all concentrations and pH.

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25
What to do if you don't get crystals?
* Check purity and stability * Remove cysteins and other trouble makers * Remove flexible parts * Try single domains * Try physiologically relevant complexes
26
What is an X-ray photon?
An elementary particle that has zero rest mass and always moves at the speed of light, a bundle of electromagnetic energy packet
27
Why do we use X-rays?
Because they have a wavelength similar to inter-atomic distances
28
What happens when a coherent monochromatic beam of X-rays is fired at a crystal?
It diffracts from the electrons in all directions
29
What happens to waves that are out of phase?
They cancel each other out
30
What happens when waves are in phases in X-ray Crystallography?
Diffraction/reflection spots can be detected
31
What is Braggs law?
For the upper and lower beans to be in phase at the detector: 2d sin(θ) = n λ
32
Reflections only occur at specific values of _
q (whole number values of n)
33
True or false: For every angle of incidence, all planes meet the Bragg law conditions
False, only a subset do Means that a crystal must be rotated to vary the angle of incidence of the beam
34
The goal of diffraction experiments is to enable what?
Constructive diffraction
35
What is resolution?
d How fine and how much detail we can see in the determined structure. Smallest spacing that will be resolved. Measured in Å
36
Although d is a variable in Bragg's equation in reality is is dictated by _
The crystal
37
True or false: If we see ‘reflections/spots’ resulting from sets of planes, then this sums the scattering of many bits of the protein.
True
38
What does the size of the diffraction spot tell us?
The amount of matter on that set of planes
39
Why are crystals usually frozen?
To protect them from radiation damage
40
What is synchrotron radiation?
Electromagnetic radiation when charged particles (electrons) are radially accelerated (moved in a circular path under vacuum)
41
In general intensities of spots decrease from the _ to the _
Centre to the edge
42
What does a dark ring on a diffraction image represent?
Probably the solvent
43
What do black spots on diffraction patterns represent?
A reflection The position and intensity of the spots are related to the electrons of each individual protein atom
44
The spacing between the spot contains information about _
The geometry of the crystal For example the dimensions of the unit cell
45
The intensity of the spots contains information about the _
Contents of the unit cells For example the distribution of the contents of the unit cell - atomic positions and properties
46
Each spot intensity needs to be matched to _
A set of planes
47
Must replace all _ reflections with the average
Equivalent Determine average intensity for equivalent reflections from the same set of planes
48
True or false: More symmetrical data is easier to analyse
True
49
What does the number of reflections tell us?
The total number of spots measured
50
What does the number of unique reflections tell us?
The number of sets of planes.
51
The more _ the higher the resolution
Reflections
52
The higher the symmetry of the space group the fewer the number of _
unique sets of planes But each set contains more information
53
True or false: Each electron will contribute to many spots on one set of planes
False Each electron will contribute to the intensity of many spots on many sets of planes.
54
What kind of analysis is used to correlate the information about how much matter there is along different parts of the unit cell?
Fourier Analysis
55
What three characteristics describe scattered waves?
Phase, wavelength, amplitude
56
In Fourier mathematics what does the strength of a diffraction spot tell us?
The waviness of the electron density
57
What is the phase problem
We cannot measure the phase of a wave, only amplitude and intensity. But we need the full wave equations to compute the molecular image by Fourier transform
58
What are the three commonly used methods for solving the phase problem?
Multiple Replacement (MR) Multi-Wavelength Anomalous Dispersion (MAD) Molecular Isomorphous Replacement (MIR)
59
When to use Multiple Replacement (MR)?
When there is an existing closely related structure
60
When to use Multi-Wavelength Anomalous Dispersion (MAD)
o If there is not a closely related structure o If there is enough methionines and suitable expression with selenomethionine as methionine source? o Or Sulphur, Zinc atoms in the protein
61
When to use Molecular Isomorphous Replacement (MIR)?
If you can soak heavy atoms into your protein crystals
62
What is the symbol for phase value?
µ
63
What is Fourier analysis?
A reversible process that can swap between the wave describing our electron density and the intensity of spots diffracted from planes
64
What is molecular replacement doing?
Taking the structure of a different protein (imagined in the same crystal type) and taking a Fourier transform of it. We can then combine the phases produced by this Fourier transform, with the amplitudes from our diffraction to do the Fourier summation to produce an image of the crystal.
65
Why is molecular replacement an iterative process?
Because the image produced will not be exactly like our crystal but will have features in it that the original phase model did not. From this, we can create a better picture of what our protein crystal looks like. We Fourier transform that to give us phases and repeat.
66
What is an electron density map?
A three-dimensional description of the electron density in a crystal structure, determined from X-ray diffraction experiments.
67
What are Structure factors?
They describe the diffracted waves from scattering planes (h,k,l)
68
True or false: The electron density map describes the contents of a single unit cell
False Describes the contents of the unit cells averaged over the whole crystal
69
How to improve phases in electron density map?
Solvent flattening or averaging
70
How to improve electron density map/model?
Refinement and rebuilding This is an iterative process.
71
Information about the finest details comes from the _-angle part of the diffraction pattern
Highest
72
Fine details can only be observed if the crystals are _
well ordered Any differences between, or motion of the crystals blurs the image
73
What is density interpreted as?
A series of atoms that follow the known atomic connections. (Protein amino acid sequence)
74
What is COOT used for?
X-ray data electron density map fitting/model building
75
What is PyMol used for?
Model viewing
76
What is solvent flattening?
It is used to clean up the model by ignoring the solvent that exists within the crystal
77
True or false: It is easy to see bound ligands, ions and water molecules
True Water molecules just need to be well ordered and resolution needs to be around ~2.5 Å
78
What is skeletonization?
Making a "bones" representation of the map
79
What do you do after making the first skeletonized model?
Turn attention to side chains Use atoms from the model to calculate phases
80
The process of model building is _
Iterative Each new model improves the phases Goes on until satisfactory
81
What is refinement?
It seeks to find the model that best predicts our original observations while simultaneously satisfying what we know about the chemical structure of proteins (such as phenyl rings are flat)
82
Why are restraints employed?
To prevent the protein from flying apart or to prevent noise. Secondary structure, hydrogen bonding, multiple copies of molecules in the unit cell etc
83
Refinement aims to minimise R. What is R?
R =∑|Fo-Fc|/(∑(Fo) Where F0 is observed amplitude (from the X-ray experiment) and Fc is calculated amplitude from the model
84
R-factor
Reliability Measures how closely the diffraction amplitudes predicted by our model matches the diffraction amplitudes we actually observed.
85
Why use difference maps?
Reduce/remove potential phase bias and errors
86
F0-Fc map
F0 (native/observed) map differs from Fc (calculated) map where there are missing or wrongly placed atoms Produces positive/negative peaks in areas where F0 differs from Fc Identifies errors in a model
87
2F0-Fc map
F0 plus a difference map F0-Fc Map should look like the corrected model Positive densities are caused by structural features not present in the model Negative densities are caused by features in the model that are not real Examine a 2F0-Fc map to guide the construction of the new model
88
Omit map
Used to reduce the bias introduced by model-calculated phases Atoms from questionable regions are omitted
89
Usual values of R-factor
20-25% Because of crystal quality and accuracy of the model phasing quality and resolution
90
How to calculate R-free
Same as calculating R but 5-10% of reflections are removed randomly from the data prior to refinement and used for this calculation
91
What is R-free?
An unbiased measure of the success of structural refinement No more than 5% higher than R in good models
92
Pros of R-free over R
More objective Not biased towards certain reflections Avoids model bias and overfitting
93
Purpose of Ramachandran plot
Should be able to define whether or not the main chain dihedral angles fall into spatially allowed conformational regions
94
B-factor
Describes to degree to which the electron density is spread out for each atom Proportional to the smearing An indicator of the thermal vibration of atoms
95
What does B-factor tell us about our model?
The true static or dynamic mobility of an atom and errors in the model building How the electron density of an atom is broadened by disorder in the crystal
96
Local static disorder
Atom positions change from one unit cell to another
97
Local dynamic disorder
Atom positions change over time during the measurement
98
B factor values
<10 is a sharp model >50 means atoms moving so much you can barely see them Higher on surface residues

Protein Structure and Analysis (9 decks)

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