Midterm: Lecture 8 Protein crystallography Flashcards
What does the diagram on slide 1 show?
A single large crystal of satellite tobacco mosaic virus
What does the second diagram show on slide 1?
TMV coat protein disk
Define protein:
A polymeric macromolecule joined together by peptide bonds
The virus can be crystallized like what?
Salt
What is TMV?
Mosaic pattern on leaf
Several methods are currently used to determine the structure of a protein, including: (3)
- X-ray crystallography
- NMR spectroscopy
- Electron microscopy
What does each method have?
Advantages and disadvantages
In each of these methods, what does the scientist do?
The scientist uses many pieces of information to create the final atomic model
Primarily, the scientist has what?
For X-ray crystallography:
For NMR spectroscopy:
In electron microscopy:
Some kind of experimental data about the structure of the molecule
this is the X-ray diffraction pattern
it is information on the Local Conformation and Distance Between Atoms that are close to one another
It is an image of the overall shape of the molecule
What is NMR spectroscopy more like?
Magnetic resonance imaging
For X-ray crystallography, this is the X-ray diffraction pattern
What does this mean?
Gives idea of how atoms are organized
X-ray crystallography:
For this method,
The protein is purified and and crystallized, then subjected to an intense beam of X-rays
The proteins in the crystal…
Diffract the X-ray beam into one or another characteristic pattern of spots, which are then analyzed (with some tricky methods to determine the phase of the X-ray wave in each spot) to determine the distribution of electrons in the protein
The resulting map of the electron density…
Is then interpreted to determine the location of each atom
What is diffraction?
Spreading of waves as they pass through or around an obstacle (ex: how light diffracts)
What happens in protein crystallography (from what I wrote)?
Purify protein, then shine beam (X-ray), and looking at diffracted pattern, can determine how atoms are organized (structure)
What does Methods for Determining Atomic Structures show? (From what I wrote)
Looking at diffraction patterns, can tell location of atoms
What can X-ray crystallography provide?
Very detailed atomic information, showing every atom in a protein or nucleic acid along with atomic details of ligands, inhibitors, ions, and other molecules that are incorporated into the crystal
However, the process of crystallization…
Is difficult and Can impose limitations on the types of proteins that may be studied by this method
What is X-ray crystallography an excellent method for?
Determining the structures of rigid proteins that form nice, ordered crystals
What are for more difficult to study?
Why?
Flexible proteins
Because crystallography relies on having many, many molecules aligned in exactly the same orientation, like a repeated pattern in wallpaper
Flexible portions of protein…
Will often be invisible in crystallographic electron density maps, since their electron density will be smeared over a large space
Q: What’s a molecular function of protein?
What does this mean?
Enzyme- changes protein confirmation
Protein confirmation: Neurotransmission
Shape: Protein shape is determined by…
Types
The amino acid sequence and how they are … binding
Primary, secondary, tertiary, quaternary
What are rigid proteins? (What I wrote)
Enzymes
Proteins that are static (ex: structural proteins)
What are flexible proteins? (From what I wrote)
Ex: proteins bound to ligands (Ex: receptor proteins)
(ion, drug) may have different configuration
Neuronal protein is what? (from what I wrote)
Neuronal protein (Rhodopsin) is flexible (different shape)
The birth of X-ray crystallography:
Lecture reported…
What did this prove?
Of the diffraction of X-rays by a crystal- the mineral zinc sulphide
That X-rays were waves
The birth of X-ray crystallography:
Results could be interpreted simply as…
What did he realize?
Arising from the reflection of X-rays by planes of atoms in the crystal
That X-ray observations, of the kind initiated by Laue, provide evidence for which the arrangement of atoms in the crystal could be inferred
What did Max Laue’s photo of X-ray diffraction from ZnS reveal?
Spots of varying shape and intensirh
What did Rosalind Franklin do in 1952?
Photograph crystallized DNA fibers
What is the distinctive “X”?
Tell-tale pattern of a helix
What does it mean if the X-ray pattern is regular?
Dimension of the helix stays the same
What does the “X” slide show?
Rosalind Franklin’s X-ray diffraction pattern
What did Frederick Wilkins do?
X-rays to produce diffraction images of DNA molecules
In X-ray diffraction pattern,
What do the horizontal bars mean?
What does the vertical distance between the bars mean?
The closer the spots, the larger the actual distance
The horizontal bars correspond to helical turns,
The vertical distance between the bars -34A degrees is a measure of the height of one turn
What does the distance of patterns mean? (From what I wrote)
Distance of patterns gives distance of height of helix
What is 3.4A?
What does this correspond to?
The distance from the middle of the X-ray pattern to the top
The distance between two stacked base pairs
Since the height of one helical repeat is… (3)
34A degrees, and the distance between stacked base pair is 3.4A, there must be 10 nucleotides per helical repeat
What is the helix’s pitch?
How can it be calculated?
Its degree of rise
From the angle the “X” makes with the horizontal axis
Fundamentals of Protein Crystallography explained:
(From what I wrote)
3 landmarks:
DNA cloning, DNA sequencing, PCR
Fundamentals of Protein Crystallography explained:
(From what I wrote)
What is DNA cloning?
Take DNA, put in plasmid DNA, bacteria will multiply- how we amplify, bacteria will manufacture the protein
Fundamentals of Protein Crystallography explained:
(From what I wrote)
Cell organelle…
Lysosome, subcellular structure (compartment)
Fundamentals of Protein Crystallography explained:
(From what I wrote)
What does dehydrate in a controlled manner mean?
Remove water
What are the 2 ends?
Why are they called that?
C and N terminals
C means carboxylic, N means amino group
