Lec 24

Question 1

For resolution of cryo EM what does red and blue mean

Answer 1

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

Question 2

What is FSC (Fourier shell correlation)

Answer 2

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

Question 3

How do you do FSC in cryo EM

Answer 3

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

Question 4

In Fourier space where is the resolution highest and lowest

Answer 4

Lowest in the middle

Higher as you move further away

Question 5

What do you look for when comparing the two structures from FSC in Fourier space

Answer 5

You look for the correlation (similarity) between the two structures

1 is 100%

0 is 0%

Question 6

What is the gold standard FSC value for measuring resolution

Answer 6

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

Question 7

What are concentric rings

Answer 7

After doing Fourier transform of the cryo em image

You get rings

The more rings you see, higher resolution of the image

Question 8

How can you tell using concentric rings and astigmatism that the image is low resolution

Answer 8

The rings would be constricted and barely visible

Question 9

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)

Answer 9

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

Question 10

Overall when using FSC what are you conalring

Answer 10

Comparing Two random halves of the data set in Fourier space through a power spectrum

Question 11

What are the limitations of using FSC for cryo EM

Answer 11

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)

Question 12

What do we have to take into account when doing FSC

Answer 12

MAKE SURE YOU LOOK AT THE RAW DATA

Question 13

What is atomic resolution

What should we see at this point

Answer 13

1 A

The spheres of the H atoms and other atoms

Question 14

What is the resolution that gives the most accurate model

Answer 14

3.5 A

Can see alpha helices and side chains clearly

But beta sheets are worse than the helices at this resolution

Question 15

At 4-6 angstroms resolution what do we see

Answer 15

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

Question 16

What can you see at 6-8 A

What about at more than 8

Answer 16

6-8 can only really see secondary structure

More than 8 is bad resolution, blobology

Question 17

I’m a sequence alignment why do we see gaps

Answer 17

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)

Question 18

In a sequence alignment, when does the score increase and when does it decrease

Answer 18

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

Question 19

What is ERK 2 and KSS1

Answer 19

ERK two is human protein kinase

kss1 is a bakers yeast protein kinase

Question 20

What Dan multiple sequence alignments tell us

Answer 20

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

Question 21

What do the numbers in a PAM matrix mean

Answer 21

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

Question 22

Why would leucine replaced valine

Ser and Thr

Asp and glu

Lys and arg

Answer 22

Both branched, hydrophobic

Both small and oh group

Both acidic and coo- group

Both basic and nh3+ group

Question 23

What codons code for

hydrophobic

Acidic

Basic

Amino acids

What does this mean

Answer 23

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.

Question 24

What amino acids are rarely replaced by others and why

Answer 24

Trp (W): unique large hydrophobic side chain

Cys (C): only one that can make disulphide bonds

Question 25

What’s an exception to cysteine being replaced by an amino acids and why

How it is helpful

Answer 25

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

Question 26

Since amino acids are more preferred in specific secondary structures, which are preferred in

Alpha helices

Beta sheets

In flexible loops

Answer 26

Longer polar and amohipathic side chains

Hydrophobic amino acids

Glycine proline (helix breaking) asn asp ser because flexible

Question 27

After doing a sequence alignments, based on the frequency table (of amino acids in specific secondary structures) what can be seen

Answer 27

Can assign regions of the sequence to specific secondary structures accurately

H is helix

C is coiled

e is beta sheet

Question 28

What is divergent evolution in proteins

Answer 28

Had common ancestors but due to speciation event or duplication their function diverged

Question 29

How can you tell that something arose from divergent evolution

Answer 29

If you compare the sequences and they have high , 40%, identity that means they are from divergent evolution

Question 30

What does it mean if the sequences are more than 40% identical

Answer 30

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)

Question 31

What is convergent evolution example

Answer 31

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