TB3-XRC Flashcards
What is SAXS used for?
Collects scattered x-rays from molecules in solution, from which nanoscale density differences in a sample can be quantified.
What is the major problem in SAXS?
The separation of the weak scattered density from the strong main beam.
What does the electron density show?
The electron density corresponding to the 3D structure of a molecule gives the probability of finding an electron at each point in space. X-rays bounce of electrons when they then hit them.
What is a diffraction pattern?
When you shine a light beam through a crystal, you get a distinctive pattern of bright spots called a diffraction pattern. This pattern is actually three dimensional – if you move the imaging planes (or rotate the crystal) you see different parts of it.
What causes diffraction patterns?
Diffraction patterns are caused by the constructive interference of light.
How are diffraction patterns related to electron density?
It turns out that the diffraction pattern is the Fourier transform of the electron density. Both the electron density and the diffraction pattern are functions of three dimensions (i.e., defined at every point in a 3D volume). Each bright spot in the diffraction pattern corresponds to one sinusoidal component of the electron density. The Fourier transform then gives a amplitude and a phase for each spot, but it’s only practical to measure the amplitude, not the phase.
What does the brightness of a spot tell us?
amplitude
What is noise?
Any deviation from what the image would ideally look like. It can be reduced with filters.
What is a convolution?
The mathematical combination of the light sources from the object with the PSF to produce the resulting image.
What are the 3 major steps in XRC?
- Crystallization
- Exposure to an intense beam of x-rays and collection of patterns of reflection
- Fourier transformations to work out the arrangement of atoms in the molecule
What is supersaturation and when is it used?
When a solution contains more than the maximum amount of solute that is capable of being dissolved at a given temperature.
It’s used to crystallize samples in XRC acting as a driving force for both crystal nucleation and growth.
What is vapor diffusion?
In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal. The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram.
What is batch crystallization?
The batch method relies on bringing the protein directly into the nucleation zone by mixing the protein with the appropriate amount of precipitant. This method is usually performed under a mixture to prevent the diffusion of water out of the drop.
What is amorphous precipitation?
when protein or precipitant are in too high a concentration one can see brown matter with no distinct shape and size
What is phase separation?
protein or detergent may separate to a different phase when mixed with certain precipitants at high concentrations
What do under-saturated crystal drops look like?
Under saturated drops, often completely clear and devoid of any precipitation.
What happens at low supersaturation?
At low supersaturation, crystals can grow faster than they nucleate, resulting in larger crystals
How is supersaturation generated?
- cooling
- evaporation
- addition of anti-solvent
Why are membrane proteins hard to crystallize?
Detergents can inhibit crystallization, so it’s difficult to obtain crystals of integral membrane proteins because detergents are required to release the proteins from the biological membrane.
What is a tesselation?
A tessellation or tiling is the covering of a surface, often a plane, using one or more geometric shapes, called tiles, with no overlaps and no gaps.
How do you name angles and faces of unit cells?
When naming angles and faces, you name based on the missing letter. E.g. if enclosed by a and b, the other face is gamma (c). If enclosed by b and c, the missing face is alpha (a).
What is synchrotron radiation?
Synchrotron radiation is emitted by charged particles travelling at relativistic speeds when they accelerate. In a synchrotron, this involves electrons being steered around a curved path by magnetic fields.
• Broad, continuous spectrum from microwaves to hard x-rays are produced
What are wigglers and undulators?
Wigglers are used in synchrotrons: the electron beam is passed through an array of magnets with alternating polarity, generating radiation at each bend in the electron path.
Undulators also oscillate the trajectory of the electron beam, but do so to generate a narrow energy band, unlike wigglers.
What is a goniometer?
Exists to position the crystal very precisely so its always in the electron beam
What is a beamstop?
Prevents the beam from hitting and damaging the detector.
Compare film to CCDs and pixel-counting detectors.
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.
What is grid scanning and why is it used?
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
What do the positions and intensities/phases of diffraction spots tell us?
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.
What does Bragg’s law state?
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.
State the equation related to Bragg’s law
nλ = 2d sinθ
What are Millers Indices?
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).
What is the reciprocal lattice?
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.
What are Argand diagrams used for?
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
What are structure factors?
Structure factors are mathematical functions that describe the amplitude and phase of a wave diffracted from crystal lattice planes, characterized by Miller indices.
What is data reduction?
When you reduce diffraction image data to a list of Miller indices with associated intensities (calculated by integration).
What is the residual of merging?
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)
What is CC-half?
measures the correlation between randomly selected half-sets of the data. Can determine resolution limits
What is I/σl – (the intensities divided by their error)?
can be considered an estimate of signal-to-noise for a dataset, but is less reliable on weaker reflections.
What is completeness?
the % of theoretically available reflections that have been observed. Should be >96%.
What is multiplicity?
how many times each reflection has been observed.
What is the phase problem?
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.
What are Direct Methods to the phase problem?
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.
Describe Patterson Methods for the phase problem
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.
What is isomorphous replacement?
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.
What is multiple isomorphous replacement?
involves repeating the experiment with a second derivative and looking for agreement with one of the first derivative solutions to solve any ambiguities.
What are Harker constructs?
Harker constructs allow us to show the two possible phases for a crystal.
What is solvent flattening?
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.
What is solvent flipping?
changes the sign of structure factors of the solvent regions, allowing the protein and solvent structure factors to be treated as statistically independent.
What is non-crystallographic symmetry?
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.
What is anomalous scattering?
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˚.
What are Friedel pairs?
Bragg reflections related by inversion through the origin.
What is Friedel’s law?
Friedel’s law therefore states that members of a Friedel pair have equal amplitude and opposite phase (Miller indices have their signs inverted).
What is single anomalous diffraction?
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).
What is multiwavelength anomalous diffraction?
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.
What is molecular replacement?
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.
What is fragment-based molecular replacement?
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
