Crystallography

Question 1

How does it give a diffraction

Answer 1

Xray scattered by the electrons of the sample
In most directions the waves interfere and there is no signal
Where there is constructive interference this leads to signal
From adjacent crystal planes
The symmetry of the pattern represents the structure symmetry

Question 2

Space groups

Answer 2

14 different space lattices
Defines the angles of the unit cells
What symmetry is present

Question 3

What does the machine look like

Answer 3

Crystal detracts the source beam
Positions, intensities and phases contain information for structure
Cannot be refocused so measured by device
3D electron density map created from Fourier transformation

Question 4

Braggs law

Answer 4

Greater angle higher res
n X wavelength = 2d sin theta
Where theta is the deflection

Question 5

Ewald sphere

Answer 5

Sphere has radius 1/lambda
Crystal sits at centre
Direct beam gives the reciprocal space
Diffraction beam gives the diffraction maximum
The distance between is the diffraction vector
Can be aligned with the planes to see which points of the lattice are in a diffraction condition

Question 6

Changing the planes in an ewald sphere

Answer 6

Straight- concentric circles

Diagonal- diffraction lunes

Question 7

Concept of resolution

Answer 7

Solvent isn’t fixed so no diffraction
Low res 5.5- peaks all merge
High res 0.8 - see the individual hydrogen atoms

Question 8

Generation of electrons

Answer 8

Windows made of beryllium foil
Cathode emits electrons which are focused by a copper anode through the window
Moving of electrons between shells generates X Ray’s
Monochromatic to allow only one type of radiation through
Crystal rotated on goniostat
Detector

Question 9

Synchrotrons

Answer 9

Circulate electrons at high speed
Emit EM radiation when change direction due to magnets
Sample destroyed in 3 uS

Question 10

Detectors

Answer 10

Scintillator (looks like satellite plate) collects the diffraction signals and filters by fibre optic taper to CCD
Larger area means faster readout
Pixel array detectors- direct photon counting, no noise

Question 11

Sample prep

Answer 11

Crystal in fibre loop
Glycerol used to prevent ice
Flash frozen

Question 12

Isomorphous replacement

Answer 12

Interference with heavy atoms
Added to unit cell in different positions (Cys)
Crystal packing can’t be altered
Intensity differences calculate atom position
Contribution to each reflection
Estimate of the phase
Compared the solutions for each heavy atom to determine phase

Question 13

Multi wave anomalous dispersion (MAD)

Answer 13

At certain freqs heavy atoms absorb radiation causing different intensities
3 data sets at different wavelengths
Effect of atoms on native intensities calculates phase
Differences are small so need synchrotron

Question 14

Molecular replacement

Answer 14

Phasing model imposed onto unit cell

Need 30% sequence identity

Question 15

Difference electron density maps

Answer 15

Fobs - Fcalc
Shows the density difference between the model and the native
Can show what has been missed

Question 16

Stages of structure based drug design

Answer 16

Cloning and protein expression
Purification (His tag, ion exchange, size exclusion)
Crystallisation (sparse matrix screening with different buffer conditions) by hanging drop
Dat collection
Structure

Question 17

Phase and intensity

Answer 17

Phase= colour
Intensity= brightness

Question 18

Refinement and R factor

Answer 18

Difference between calculated and observed amplitudes
General target 20%
R free is the same but for a reflection subset
Difference between should be 10%

Question 19

B values

Answer 19

Measure of the thermal motion
Causes blurring of each atom
Depends on the rigidity of that section
Data collection in liquid nitrogen helps reduce B values