Lec 24 Flashcards
For resolution of cryo EM what does red and blue mean
Red means more variable due to
flexibility in the structure
Or because certain domains are being averaged out because some are present and some missing in the protein (due to truncation)
Blue is more ordered and defined regions
What is FSC (Fourier shell correlation)
A method to measure the similarity between two 3D maps of a protein over different spatial frequencies (in Fourier space)
Way to find the resolution of the model
How do you do FSC in cryo EM
You take the final structure
Take the number of particles that were uses to make that structure (ex. 50000)
Split the particles randomly in two to make two independent 3D volumes
Redo the Fourier transform and then compare the two structures independently in Fourier space
In Fourier space where is the resolution highest and lowest
Lowest in the middle
Higher as you move further away
What do you look for when comparing the two structures from FSC in Fourier space
You look for the correlation (similarity) between the two structures
1 is 100%
0 is 0%
What is the gold standard FSC value for measuring resolution
0.143
So this is where there is good correlation between what resolution you see and what the resolution should be
This is the value that tell you your resolution
What are concentric rings
After doing Fourier transform of the cryo em image
You get rings
The more rings you see, higher resolution of the image
How can you tell using concentric rings and astigmatism that the image is low resolution
The rings would be constricted and barely visible
On a FSC vs resolution plot at low resolution (high number) what happens to the correlation (FSC)
Then what happens as resolution gets higher (lower numbers)
At low resolution everything correlates so you have a high FSC
As resolution increases and you see secondary structure, the correlation falls to the 0.143 value
Then becomes noise
At the 0.143 we’d say the OVERALL resolution of the structure is the one shown at 0.143
Overall when using FSC what are you conalring
Comparing Two random halves of the data set in Fourier space through a power spectrum
What are the limitations of using FSC for cryo EM
It depends on the quality and quantity of the data and Is affected by noise, motion, alignment errors, and sample heterogeneity
Could over or under estimate the resolution depending on how you analyze the data
Might not provide enough info to get accurate models at medium resolution (if resolution more than 4 angstroms your good)
What do we have to take into account when doing FSC
MAKE SURE YOU LOOK AT THE RAW DATA
What is atomic resolution
What should we see at this point
1 A
The spheres of the H atoms and other atoms
What is the resolution that gives the most accurate model
3.5 A
Can see alpha helices and side chains clearly
But beta sheets are worse than the helices at this resolution
At 4-6 angstroms resolution what do we see
Starts to get blurry
Can still see the bulky side chains
But small side chains and flexible side chains like arg and lys are invisible
What can you see at 6-8 A
What about at more than 8
6-8 can only really see secondary structure
More than 8 is bad resolution, blobology
I’m a sequence alignment why do we see gaps
That region of the protien is constantly mutating and deleting and not playing an important role
These regions are unstructured flexible loops (not part of the core domain of the protien)
In a sequence alignment, when does the score increase and when does it decrease
Increase When the alignment is making identical and conservative matches
Decrease when it’s need to introduce gaps to being the regions of identical residues
What is ERK 2 and KSS1
ERK two is human protein kinase
kss1 is a bakers yeast protein kinase
What Dan multiple sequence alignments tell us
It’s can show conserved sequences between many organisms which can show that it is important to the function of the protien
Ex. Wee1 doesn’t have the conserved kinase sequence meaning it doesn’t work as a kinase
What do the numbers in a PAM matrix mean
The frequency that one amino acid gets substituted/mutated for another
Ex. A big number for alanine and glycine means that alanine is often replaced by glycine
Why would leucine replaced valine
Ser and Thr
Asp and glu
Lys and arg
Both branched, hydrophobic
Both small and oh group
Both acidic and coo- group
Both basic and nh3+ group
What codons code for
hydrophobic
Acidic
Basic
Amino acids
What does this mean
xUx
GAx
CGx, AA(AG), AG(AG) (means A or G)
Even though mutations occur, the end result will be a similar amino acid (ex. Hydrophobic to hydrophobic) which leads to less damage.
What amino acids are rarely replaced by others and why
Trp (W): unique large hydrophobic side chain
Cys (C): only one that can make disulphide bonds
What’s an exception to cysteine being replaced by an amino acids and why
How it is helpful
Cysteine to serine: because they are both the same size and have one polar atom (O vs S)
Helpful in purification because if you mutate it to serine, don’t need to use BME/reducing agents in the purification
Since amino acids are more preferred in specific secondary structures, which are preferred in
Alpha helices
Beta sheets
In flexible loops
Longer polar and amohipathic side chains
Hydrophobic amino acids
Glycine proline (helix breaking) asn asp ser because flexible
After doing a sequence alignments, based on the frequency table (of amino acids in specific secondary structures) what can be seen
Can assign regions of the sequence to specific secondary structures accurately
H is helix
C is coiled
e is beta sheet
What is divergent evolution in proteins
Had common ancestors but due to speciation event or duplication their function diverged
How can you tell that something arose from divergent evolution
If you compare the sequences and they have high , 40%, identity that means they are from divergent evolution
What does it mean if the sequences are more than 40% identical
The secondary structures are conserved, but the length of the helices and beta strands are diff
Most of the core hydrophobic residues are conserved, but not all of them are identical (like ile to leu)
The loops regions are mutating very fast and are more likely to change than the regular secondary structure regions
The surface residues are more likely to change than the core hydrophobic ones (the further in are more important, less mutated)
What is convergent evolution example
The structure arose independently but developed similar function
Ex. The catalytic triad of the subtilisin and chemotrypsin proteases
They look diff but do the same thing using same mechanism
