Fan Lecture 6 Flashcards
What is SIRAS?
Single Isomorphous Replacement with Anomalous scattering
Describe the steps of SIRAS.
1) High-energy electrons emitted from the cathode collide with the copper atoms in the anode, they eject k-shell electrons from the copper atoms
2) Ejection of k-shell creates vacancies and electrons from higher energy orbitals transition to fill these vacancies.
3) When they transition, they release energy in the form of x-rays. These x-rays are characteristic of the element involved. The energy of the emitted x-rays corresponds to the energy difference between the first and last electron orbitals involved.
4) The emitted x-rays can be detected using appropriate detectors.
What is coherent scattering?
same frequency but with a 180-degree phase shift
- occurs for all atoms that do not absorb the x-rays
What is anomalous scattering?
phase change from scattering that depends on atomic number but is not 180 degree
- occurs for all atoms that absorb x-rays
How is x-ray generated in the laboratory?
High energy electrons from the cathode eject K-shell electrons in the anode. X-rays are produced when electrons transition from higher energy orbitals in the copper fall to the vacated k-shell
- producing K-alpha and K-beta x-rays respectively
How do you know which filter to use to get a single wavelength x-ray, given metal M as anode?
1) Determine desired wavelength of the x-ray you want to isolate
2) Filter must absorb x-rays with wavelengths other than the desired one.
3) Consider thickness, thicker filters tend to absorb more x-rays, while thinner filters allow more x-rays to pass through
4) Absorption edge of the filter should match the energy of the undesired x-rays to ensure efficient absorption of these x-rays
Ex. A filter of Ni removes the Cu-K-beta radiation, leaving nearly monochromatic Cu-K-alpha radiation.
- Nickel has absorption edges at energies higher than the Cu-K-alpha radiation but lower than the Cu-K-beta radiation
What is Friedal law?
The equivalence of the intensity of each reflection hkl is identical to the intensity of reflection -(hkl).
When is Friedel law not true and why? Explain it in complex coordinates.
Anomalous scattering: introduces additional phase shifts that also affect the relationship between structure factors and their complex conjugates
- Fph2 = Fph1 + ΔFr + ΔFi
ΔFr and ΔFi are also reflected to each other on the real axis thus affecting the normal reflection.
What is MAD?
Multiwavelength Anomalous Dispersion
Describe the steps of MAD.
1) Obtain well-ordered protein crystals suitable for x-ray diffraction studies. Replace sulfur atoms for selenium if the proteins lack native heavy atoms.
- overexpress the protein in a host bacterium that requires methionine for growth and substituting Se-methionine for methionine in the growth medium
2) Collect x-ray diffraction data from protein crystals at 3 or more wavelengths: at, below, and above the absorption edge of the heavy atom.
- rotate the crystal in the x-ray beam and recording the intensity of diffracted x-rays at various angles and wavelengths
3) analyze x-ray diffraction data to identify anomalous scattering signals corresponding to the presence of heavy atoms in the crystal lattice. Measure the difference in structure factors between reflections at different wavelengths.
What is the advantages of MAD vs MIR?
1) Higher resolution phasing, especially when anomalous scattering data are collected at multiple wavelengths.
2) No heavy atom soaking, making it suitable for proteins lacking native heavy atoms
3) Can substitute sulfur for selenium in methionine residues, simplifying heavy atom incorporation
4) Yields stronger anomalous scattering signals by optimizing data collection at wavelengths near the absorption edge of the heavy atom
5) Better success rate in solving crystal structures
What is the limitations of MAD?
1) Needs access to synchrotron facilities with tunable x-ray beams
2) Requires high-quality crystals to collect high-resolution data at multiple wavelengths
3) Costly and time-consuming
What is Fo map?
Initial crude electron density map obtained with the observed intensity data
- best case: shows continuous chains of electron density, alpha-helices, and side chains
- Worst case: not informative, more phasing or another heavy atom derivative is needed
What does positive and negative peaks mean in Fo-Fc map?
Positive peak means that the real unit cell contains more electron density at this location than implied by the molecular model.
Negative peak means that the real unit cell contains less electron density at this location than implied by the molecular model.
What is 2Fo-Fc map?
Represents the difference between observed and calculated structure factors & highlights atomic positions by subtracting background density
- used to identify atomic positions, assess model quality, and detect erros
