TB3 - XRC Flashcards

Question

What is a beamstop?

Answer 1

Prevents the beam from hitting and damaging the detector.

Answer 2

Historically, photographic film was used. However, this is slow, not precise and very labor intensive. We now use CCD (charge-coupled devices) which are fast with a high dynamic range and low noise. These need to be run at low temperatures and corrected for errors. Pixel counting detectors is a new type of technology, whereby each pixel on the detector counts photon events independently. These detectors are incredibly fast, allowing for constant rotation of the crystal and no more stopping for detector readouts, as well as having a very high dynamic range.

Answer 3

Grid scanning: crystal mount is translated, and short exposures are taken of many areas of the sample. It can help with: • Locating small crystals and those in opaque surroundings • Identifying regions of well-diffracting and poorly-diffracting crystals • Centering of samples where optical centering is difficult

Answer 4

The position of a diffracted spot depends on the shape and size of the unit cell, whilst intensity and phase of the spot depends on the structure of the molecule.

Answer 5

Bragg’s law states that when the x-ray is incident onto a crystal surface, its angle of incidence will reflect back with the same angle of scattering. And, when the path difference, d, is equal to a whole number, n, of wavelength, a constructive interference will occur.

Answer 6

nλ = 2d sinθ

Answer 7

These are a group of three numbers (h, k, l) that indicates the orientation of a plane of atoms in a crystal. Higher Miller indices indicate finer sampling (small d between planes) leading to a greater scattering angle and higher resolution (based on Bragg’s Law).

Answer 8

In a crystal, the atoms form a periodic array, and the corresponding diffraction pattern on the detector is also a regular array of light spots. This is the reciprocal lattice, e.g., if atoms are more widely spaced, the light spots get closer. Yet, when atoms get closer in one direction only, the reciprocal lattice expands in this direction only.

Answer 9

These are tools for allowing us to add waves together considering the wave properties rather than atomic properties. - To add them together, you add nose-to-tail - Phase is the angle going anti-clockwise around the circle - Amplitude is the length of the vector away from the origin - NB. Experiments are monochromatic, so all waves have the same wavelength

Answer 10

Structure factors are mathematical functions that describe the amplitude and phase of a wave diffracted from crystal lattice planes, characterized by Miller indices.

Answer 11

When you reduce diffraction image data to a list of Miller indices with associated intensities (calculated by integration).

Answer 12

When undergoing data reduction, each reflection will be observed more than once in a data set. We merge these together to form a unique reflection list. How well these observations agree with each other when they’re merged gives an estimate of the quality of the data. • A lower residual on merging indicates better data (generally <0.1 for an entire dataset)

Answer 13

measures the correlation between randomly selected half-sets of the data. Can determine resolution limits

Answer 14

can be considered an estimate of signal-to-noise for a dataset, but is less reliable on weaker reflections.

Answer 15

the % of theoretically available reflections that have been observed. Should be >96%.

Answer 16

how many times each reflection has been observed.

Answer 17

It is only possible to measure the amplitude of the diffraction pattern spots by experimental means; the phase information is missing. Without phase information, it is impossible to reconstruct the electron density in the unit cell.

Answer 18

These use the relationship between known amplitudes and phases to predict phases for small molecules. It requires high resolution to work, hence why it is often non-applicable to larger molecules like proteins.

Answer 19

While the phases determine the positions of the peaks of electron density across the unit cell, and therefore the positions of the atoms, the presence of a strong diffraction spot alone gives a strong indication that features must be present with the corresponding spacing. Thus, the structure factor magnitudes alone contain information about the spacing of atoms in the structure. This information may be accessed by calculation of the Patterson function, determined via a map that uses the squared structure factor magnitudes, and all the phases set to 0. Instead of peaks at the atomic positions, the Patterson map shows peaks at every position that corresponds to an interatomic vector in the structure. To do this, you need a simple structure to be able to identify positions of a small number of atoms – from this you can estimate the phases for all reflections and bootstrap you way up.

Answer 20

Isomorphous replacement compares the diffraction data from a native protein crystal (nothing bound) to one where heavy atoms have been bound. The Patterson function is then used to calculate the positions of the heavy atoms.

Answer 21

involves repeating the experiment with a second derivative and looking for agreement with one of the first derivative solutions to solve any ambiguities.

Answer 22

Harker constructs allow us to show the two possible phases for a crystal.

Answer 23

A protein crystal can contain a high amount of solvent, causing space that doesn’t contribute to scattering. To remove the noise caused by the solvent, the electron density is set to a low constant value in these regions. This sharpens the protein density and gives better maps. You would then recalculate a new set of phases.

Answer 24

changes the sign of structure factors of the solvent regions, allowing the protein and solvent structure factors to be treated as statistically independent.

Answer 25

If a protein has more than one molecule in the asymmetric unit, you can use NCS. By calculating the relationship between the molecules in the ASU, it is possible to average the electron density of the molecules. This creates a better signal-to-noise ratio and therefore more defined electron density for the individual molecules.

Answer 26

When an incident photon has high enough energy, the scattered photons gain an imaginary component to their phase (f') i.e., they're retarded compared to a normally scattered photon. Anomalous scattering uses heavy atoms to look at these properties. When you add a heavier atom, you add an extra component to the scattering, but these are still symmetrical. Thus, the amplitude is the same, but the phase is inverted. Under anomalous scattering conditions, the phase is lagged by the imaginary f’ component and so is always different by 90˚.

Answer 27

Bragg reflections related by inversion through the origin.

Answer 28

Friedel’s law therefore states that members of a Friedel pair have equal amplitude and opposite phase (Miller indices have their signs inverted).

Answer 29

Collect a single dataset with the maximum anomalous signal. This arises in two phase solutions. To determine the phase, you combine the phasing experiment for isomorphous replacement with SAD phasing, known as SIRAS (single isomorphous replacement anomalous dispersion).

Answer 30

If data is collected for a MAD experiment, you can add the additional wavelengths used onto the Argand diagram in the same way as MIR.

Answer 31

An alternative approach to the phase problem may be used when the molecule under study is similar to another molecule whose structure is already known. In this case, the method allows phases to be obtained from the known structure.

Answer 32

Fragment-based molecular replacement is now being used as we know what secondary structure elements look like and can use these as search models even in the absence of a search model with reasonable sequence homology