Midterm 3 Flashcards
(31 cards)
Difference between x-ray diffraction and neutron diffraction.
X-ray diffraction- electron density causes scattering of particles
Neutron diffraction– nuclei cause diffraction of neutrons.
ND can detect hydrogens because they have nuclei, but very little electron density.
Disadvantages of neutron diff.
Large crystals required, need high intensity monochromatic neutron source, long data collection times required since most of the neutrons pass through without scattering due to weak interaction.
Cryo-EM
A type of Transmission Electron Microscopy in which an electron beam is passed through a sample and a projection generated.
Cryo-EM uses what kind of ice?
Vitrified (glass-like); obtained by flash freezing water– prevents damage to biomolecules being studied (bc normal ice has unit cells)
Simulated annealing
A process in which a protein structure is used to fit something into a mold (like the electron density map)
How do you determine structure from a Cryo-EM image?
Many thousand of projections of the molecule are averaged together to create a high-quality of image with a resolution close to that of x-ray crystallography.
Why are stronger magnets better for NMR?
1) Resolution– separation of peaks is proportional to field
2) Specificity–
amplitude of peaks is proportional to field
What is NOE and what does it measure?
nuclear Overhauser effect– irradiating at a frequency of one peak can cause other peaks to lose intensity, those are the nearby protons; depends on the distance betweeen nuclei (larger NOE is shorter distance)
What is the assignment problem?
When you observe a strong NOE shift, you know two protons are close together, but which one?
Limitations of X-ray diffraction vs. NMR
X-ray: need crystals
NMR: can’t use on a protein larger than 40kDa (too complex), can get slightly more info on dynamics of bonds
Can you use zero nuclear spin atoms for NMR?
NO! O16 and other atoms with zero nuclear spin do not resonate, you use simulated annealing to figure out the placement of these.
IR waves are how long?
700nm to microns
What are good IR absorbers?
methane, water, ozone, CO2
Why is Raman scattering superior in protein analysis?
Water is a good IR absorber, but Raman spec doesn’t use IR so can detect proteins in solution
Asymmetric vibrations are good for
absorbing IR
Stokes Raman band and anti-Stokes Raman band
S-R: v0 - vvib
AS-R: v0+vvib
Example of something that can be learned from a single molecule study but not from an ensemble method
Different folding pathways of a protein
Several unique structural states (as in ATP synthase)
What are optical tweezers good for?
measuring energy required to unfold a molecule.
What are the two counteracting forces in an optical tweezer setup?
Scattering force– force of photons hitting the bead,
Gradient force– force of refraction of photons on the bead.
What is the size of a bead used for optical tweezers?
Micron
Concept of circular dichroism
Chiral molecules such as proteins rotate left anded and right handed CD light differently, The difference in extinction coefficient is used.
ab initio vs empirical methods
ab initio– figuring out tertiary strcture from AA sequence alone
empirical– using some prior knowledge, e.g. similar proteins, conserved regions to determine structure.
Discrepancies between CD and crystal struct.
CD is an ensemble method in solution, perhaps certain structure exist in folded and unfolded states in solution while certain forms are stabilized by the crystal.
A quick way to compare structures of a wild type v. mutant enzyme
Look at CD spectrum of both– if they match, mutation did not cause protein to unfold
