Comprehensive Guide to Recombinant Protein Expression and Structural Biology

here is a whirlwind overview of recombinant protein expression and purification techniques as well as

structural biology techniques so basically what do scientists do if they want to study what a protein looks like

and how it works how do they get cells to make or Express that protein how do they purify the protein so using protein

chromatography methods and then how do they actually use structural biology methods like X-ray crystallography and

cryoem in order to figure out what the protein looks like and then how can they do biochemical assays and stuff to

figure out how what it looks like corresponds to how it functions so the connection between Form and Function the

heart of structural biology going back to our essential Dogma molecular biology we know that the instructions for making

a protein are written in the form of a DNA Gene that then gets transcribed to make a messenger RNA copy that gets

translated to make a protein because of the universal nature of the genetic code we can stick that same

instructions from that protein into any type of cells and hopefully get them to make the right protein

a little technical detail is that when you go from a gene to an RNA initially when you transcribe it you kind of

transcribe a bunch of information that isn't needed by the ribosomes it's not needed to make the protein

um it's extra information like regulatory information introns things like this and so what happens is you get

some editing to get a mature messenger RNA and so what we put into cells if we want them to get a make a protein is

actually a DNA version of this messenger RNA which we call a cdna or complementary DNA we often just refer to

this as a gene but technically it's not a gene it's a cdna we can then stick the cdna into a vector

so a vector is going to serve as a vehicle for getting this these genetic instructions into cells often we're

using some sort of plasmid if we're working with bacteria we can take a circular piece of DNA called a plasmic

and stick it into bacteria and then bacteria can make the make the protein for us we can also use things like

Pac-Mans to make bacular viruses for insect cell expression we can use other types of like adenoviruses and things

like this if we want to express them a million cells so there's a bunch of different options depending on what type

of cells you're going to actually use and we'll get into that in a minute what type of cells but not the details about

how you go about using each of these types we call this recombinant DNA or protein

um basically when we're recombining that cdna with that with the vector we call this recombinants because we're

recombining it um and the way that we recombine it is using molecular cloning strategy so this

can include things like restriction enzymes or PCR based methods um various strategies for this cloning

and so on cloning we'll talk about cloning um in Biochemistry we're typically not talking about things like

Dolly the sheep we're just talking about moving pieces of genetic information from one place to another place to do

things like make them easier to work with or get cells to make the protein for a protein for us

speaking of those cells so I'm not going to go into the details about how we actually go about using all these

different cell types I just want you to know that there are different cell types that can be used to express proteins and

they have different pros and cons um I want you to be familiar with these so that when they show up in the papers

and stuff we're going to read you'll be you'll be familiar with them and know why the scientists might have chosen one

method or another often what you'll see is proteins will be expressed in bacteria

um as I mentioned we can use those plasmids to express things in bacteria and what's really nice about bacteria is

we can actually do like inducible expression where basically we can get these bacteria to make a protein for us

on demand so if you see a paper when they're talking about like adding ipkg basically what that's doing is it's

going to de-repress the um the protein it's going to make the cells start making that protein on demands

and that if you're interested in this I have posts on that with much much more information I just want you to know that

if you come across that that's probably what it's talking about so why use bacteria well it's cheap and

easy um why not use bacteria well if you're wanting to express like a human protein

these bacteria might not be the best option because they're going to have different post-translational

modifications we often abbreviate these as ptms when we talk about ptms we might be talking about things like

we'll talk about ptms they might be talking about things like phosphorylation

so that's the addition of those phosphate groups um glycosylation

so that's the addition of sugar chains um things like this they also have different chaperones so these

protein folding helpers so the proteins might fold differently in the bacterial cells they might not

fold well the cells might not even want to make you the protein well um and so basically there's different

conchs these are often good often good for small proteins good for small simple proteins

but they can have trouble when you're dealing with bigger things or you or you have post-translational modifications

you want to care about in those cases you might want to choose something that's more um that's more

mammalian-like or human-like or at least more eukaryotic and so the next step up is kind of like yeast cells these are

again going to be cheap and easy-ish but they have some cons they're hard to lice and they're still very different from

animal cells one another method that's commonly used is going to be insect cells or the

vacuole virus system so you might see things like sf9 that's the cell line um or you might see things like high

five to things like sf21 and you don't need to know all these names

um just if you if they show up that's what this is referring to so I used insect cell expression a lot

in my um grad school days basically it's more similar to us in terms of its modifications and chaperones it's easily

manipulable and it's easiest to scale up but it is a lot of work to maintain and it's more expensive than those bacteria

as to how it works basically you can use bacteria to make a virus that can only infect insect cells and then you infect

those insect cells and get them to make a lot of your protein so it's a cool system works well it's helpful that you

can do a lot of the work in the bacteria but then you have to switch to the insect cells

um and there's a lot of maintenance not as much maintenance though as the mammalian cells so for the mammalian

cells we're dealing with things like um Joe sells Chinese hamster ovary XB freestyle 293 which are GK cells these

are going to be most similar to humans so they're going to have similar modifications similar chaperones Etc but

they're going to be very pricey um and high maintenance and things like this um and to actually Express from those

then you typically have some sort of um you have to transfect them with various lipid reagents or use a viral

Vector system there's various ways um but basically know that those are going to be the most human-like the most

expensive um and that sort of thing so again you don't need to know the details of all

these just when we're doing our paper reading discussions I want you to be able to

um to recognize when these show up in your paper so that you're knowing what where to go to find more information but

you're not going to be responsible for for knowing for knowing the names of all of these different things this is just

for for the paper discussions things another thing that's going to show up in your paper discussions is you might see

codon usage optimization um basically there's different codons basically amino acid

um the amino acid transfers that come and connect the read the messenger RNA and bring in the corresponding amino

acid there are multiple ones that can kind of do the same job and the cells might have more than one than the other

and so you can basically there are strategies to optimize the codon usage for different cell types again you don't

need to know the details of all of this but it is going to show up in some of your papers and so this is what it's

referring to and then you can go find out more if you're interested okay so whatever the method you've

somehow gotten cells to make yours to make your protein you can also be purifying protein like native protein so

protein just out of the cells naturally make and so this is what you'll be doing with your lysozyme you're going to be

purifying it out purifying the native lysozyme out of the out of the egg whites but in most cases if we want to

study a protein we're doing it with this recombinant way and one of the benefits of this is that we can add a little

extra protein onto the end that we call a tag which will help make purification easier

so remember that because we're putting in the genetic instructions if we add a little bit more DNA on the ends well now

we're going to get a little more protein on the ends and if we get that a little more protein on the end and then we can

use that as like an affinity tag I'm basically a way where we can capture the protein wash everything off the way and

then um and then alute or push off our protein in a purified form so

then what we do is we get these proteins to make we get the cells to make our protein we break the cells open we

separate the soluble stuff from the insoluble stuff and then we can use various forms of protein chromatography

in order to purify our protein basically with this chromatography we have these columns which can which are filled with

these resin these little beads and these beads have different properties that are going to interact differently with

different proteins and in this way we're able to run a solution containing proteins through them the different

proteins will interact differently so they'll get stuck on the column or they won't get stuck on the column or they'll

go slower through the column or they'll go faster through the column and in this way we're able to separate proteins to

isolate the wand that we want we'll talk a lot more about chromatography in a minute but first I

just want to step back and say something about um about one more one more detail about

the expression and the purification strategy in those cases what we're going to be

dealing with is we're going to be dealing with water-soluble proteins but in some cases including in the

examples we're going to use some of the examples we're going to use you're dealing with a membrane protein now

membrane proteins are more complicated because when you go and you break open those cells well the membrane proteins

are going to be in with the membrane bits um and so you wouldn't want to then go

throw away that pellet and use the super the liquid on top instead what you have to do is you have to actually isolate

the proteins from that membrane one way though is commonly used is to use detergent

so basically the lipid that the membrane proteins are embedded in it had its amphiphilic it has both hydrophilic

parts and hydrophobic parts so it's got hydrophobic tails and hydrophilic heads and what happens is these phospholipids

they're going to form these by layer membranes that the protein will be embedded in

another type of ampophilic molecule is a detergent which is basically just an artificial soap if you add a detergent

to water well instead of forming those bilayer membranes it actually forms these like single layer bubble like

micelles and it does this because they have a different shape so the phospholipids these are more going to be

like rectangular so they can't form of myself very easily but they can form those bilayers whereas a detergent here

you're more like conical and so you can form these micelles so you can use a detergent because it's

still that amphipipilic nature it can kind of get into um Wiggle its way into that phospholipid

membrane and it will disrupt it and then if you have enough of the detergent it will actually make my cells that will

incorporate um incorporate the membrane protein into them

so that's one strategy you can use to actually purify the membrane proteins the whole length thing in their original

form you can also do things like just Express parts of it so basically if you look at

a membrane protein it often has three different parts for when I'm talking about a membrane protein that actually

goes all the way through so when we talk about membrane proteins we'll see that there's some that go all the way through

there's some that kind of just hang out on one side or the other um and things like this

we can talk about these proteins as having different domains so remember our domains are like our rooms in our house

um and so basically there are several different rooms in in a membrane protein you can have the Ecto

domain and so remember the Ecto domain this is going to be facing the outside of the cell

um Ecto outside um and so then you have the transmembrane domain the trans through

is going through the membrane and then you have the Endo domain so the part that is actually inside of the cell

um so this is going to be inside of the cell and outside of the cell now if you think about what's inside the cell and

what's outside the cell well outside the here you're going to have water and watery environment over here and then

you're going to have a lipid environment in the middle so the part of the protein that goes through that lipid is going to

be hydrophobic but the parts on the outsides and on the inside um like of the cell are going to be

hydrophilic when cell when proteins are hydrophilic well then we can then purify them just

like normal soluble proteins so what we can do is because we're actually we can manipulate that DNA

sequence we can manipulate the genetic instructions we can actually just Express those domains separately

so alternative Express only

Ecto or Endo domain so of course when you do this well now you're losing a lot of the

protein you're losing a lot of the information um but if you're doing things like

studying binding to the Ecto domain then this can be an okay strategy in a lot of cases

okay so going back to our story we basically we're dealing with a recombinant protein we've stuck it in

we've added a little bit extra onto the end to serve as a tag and now we're going to go and purify it

and again we're going to purify it using chromatography so when we do chromatography you'll see

a couple of different methods used so we can either be doing things in like gravity flow

so gravity flow where you basically pack your own columns so you fill these columns with

resin and there's all sorts of different size columns you can use or we can use a machine so commonly this like this is

like an actor that's just like a brand name um and here it's actually going to use

pumps to push the liquid through on through the columns again these columns are going to be

filled with different types of resin so this resin remember these are just like little beads

with different features and so there's kind of like three main types that we'll be talking about we'll be talking about

Affinity chromatography ion exchange chromatography and size exclusion chromatography which are all going to

take advantage of different things about different proteins let's take them one by one

with affinity chromatography we're going to be separating based on a specific feature such as a tag with ion exchange

chromatography we're going to be separating based on charge and with size exclusion chromatography we're going to

be separating proteins based on their size so we'll go into each of these in a little more detail but know that often

these techniques are kind of done in um done in series so that we can get a protein really really pure so how many

different purification methods you use is going to depend on how pure you need your protein to be if we're dealing with

things like structural biology where we're once really really pure proteins then we often do multiple column

chromatography steps in a row often like we'll do Affinity chromatography to get rid of most stuff then ion exchange

chromatography and then size exclusion chromatography or gel filtration and that will get us get us hopefully very

very pure protein but often we'll just be doing um for just like the quick and easy dirt quick and dirty method we'll

be doing Affinity chromatography and so let's start there the reason why we want to start with

affinity chromatography is basically it's going to be the most specific so Affinity chromatography what happens

here is that your protein has some specific feature that's kind of unique to it and so most classically this is

going to be one of those um one of those protein tags those Affinity tags that we stick on to our protein when we're when

we're doing that recombinant expression when we're doing that cloning we just stick a few extra amino acids onto the

end and then we can use resin that has a um that has a group attached to it that can actually bind specifically to that

specific sequence so because lots of people put the same specific sequence onto their protein

these companies will then sell this stuff cheaply where you're relatively cheaply still pretty expensive

um but it's cheaper than having to kind of design something that would bind to every single different protein if you

can design something to bind to that little tag that people add onto their protein and so a couple of the common

tags are going to be like a hiss tag or a strep tag and then these Affinity chromatography

beads they'll have kind of like the matching root for that now what's going to happen is when you

flow your protein with that tag through the um through the column your protein is

going to stick but that other stuff is not and so then you can kind of wash all that other stuff off while your protein

is stuck to the column and then you can add a competitor to kind of compete off your protein so you add something that

looks like the tag or the tag itself or whatever um and then this will push off your

protein and allow you to get a pure protein in the absence of all of that stuff that you washed off

so again a couple examples are his tag so a hiss tag is just six to eight histidines in a row and it's going to

bind metal coated regisons um so some often what you'll see is you'll see like nickel NTA

um and things like this you might also see like Talon which is I think that's that one is um Cobalt there's various

different metals that are used and so histidine we'll look at later is going to be very good at coordinating Metals

um and so histidine if you put a bunch of them in a row it's going to be able to bind to

bind to that column then what you do is basically once it's bound then you wash all that other stuff

off and then you push it off with this in this case amidazole which is going to act as a competitor and a midazol is

basically just that side chain part of histidine another one you'll come across is a

struct tag this is going to bind streptacting resin which is the streptavid mimic

um in the same place as Biotin and dusta biotin so basically what it is there's a super strong interaction between Biotin

and avidin and basically strepto or and streptavidin and so the the strep tab is basically a biotin mimic that's going to

bind to it well I mean it's obviously biotin is kind of like this um organic this small organic molecule

um and we're dealing with the protein sequence but they're able to bind to the same um strepto Avenue mimic in the same

place and therefore you can use this struct and resin which mimics that strip dividend in order to bind to a strep

tagged protein and then you can compete it off with this biobiotin you don't need to know all these details

you just need to know the core idea that you can add an affinity tag to your protein and then use Affinity

chromatography to specifically capture your protein wash away the other stuff and then compete off your protein

often these Affinity tags they actually when you put them on your protein you add a little bit of extra sequins in

between the tag and your protein that serves as a protease recognition sequence so when we talk about a

protease that's going to be a protein cutter some proteases we don't like because

they're just going to chew up our proteins when we're working with them but these ones are going to be

site-specific and so they're going to recognize that specific sequence and then we can cut off our protein

um and so or cut the tag away from our protein with something like a really small tag you might need not not need to

worry about cutting that tag off because it might not interfere but sometimes we're dealing with a bigger tag

something like GST which is actually like a whole protein fused onto the end of your protein

um and so this could interfere with further things and so then you're often going to cut it off

of course once you've cut it off well now you don't have that specific feature about your protein to take advantage of

so you have to charge your natural properties about the protein one of the ways that you can do this is

Ion exchange chromatography because well all proteins are going to have some sort of charge all proteins have a different

combination of amino acids and as we've seen different amino acids can have different charges and this will all

depend on the pH so remember that the pi that's the isoelectric point that's the point at

which a multi-protic molecule is net neutral and so when we talk about A protein that is definitely counts as one

of those multi-protic molecules and so it's going to have this Pi this point at which it's net neutral

and why this matters is because well if we're at a lower pH than that remember there's more protons and so this means

that we're going to have a positive charge overall for our protein and if we're at a pH that is higher than RPI

well then our protein is going to be negatively charged so when you're at a pH that is below the pi

well then what's going to happen is your protein is going to be positively charged

but if you're at a pH that's above the pi your protein is going to be negatively charged

and well we know that opposite charges attract one another so if we have a positively charged protein we can get it

to stick to negatively charged resin and if we have negatively charged protein we can get it to stick to positively

charged resin when we talk about something that's positively charged we're talking about a

cation and we talk about something that's negatively charged we're talking about an anion

so what we can do is we can use methods called kava on Exchange and anion Exchange in order to separate proteins

based on being positive or negatively charged if we have a positively charged protein

we're going to want negative resin when this isn't a strategy called cation Exchange

The Exchange is because we're going to be exchanging salt cation so like sodium ions for your protein and then

exchanging back off for insults once again and so with cation exchange your protein

is cationic you want the opposite for it and the resin is going to be negatively charged or antibiotic with anion

exchange your protein is anionic so it's negatively charged and you're dealing with positively charged resonant in this

case what you're going to be exchanging is going to be exchanging like the chloride ions or whatever the anion is

in the salt now what's going to happen is that your protein is going to be able to displace

the salt and binds but if you add more and more salt well then your protein is going to get

um your protein is going to kind of be competed off similarly to with affinity chromatography except here we're

competing it off with salt molecules you can also get your protein to come off by changing the charge of the

protein by changing the pH because remember that the charge is going to be dependent on the pH but typically we're

using salts because well if you change the pH the protein might be mad at you um and things like this often what we're

going to do is we're going to use a salt gradient so when we do it with our lysozyme we're basically we're going to

be doing ion exchange chromatography but we're going to be doing it stepwise um so basically go from low salt to

really high salt um because it's easier and we don't have to use a machine to do the gradient but

using the gradients is nice because then you can kind of separate things based on their charge rather than kind of I mean

like separate things more granularly based on their charge rather than kind of things that don't bought things that

are not very charged or in things that are really charged or I mean charged in that direction

um and so yeah so that would be your Ion exchange chromatography another method that we often use is size

exchange chromatography um so this is AKA gel filtration and so we often also abbreviate this sec

um and basically this is going to separate proteins based on their size it's often used as a sort of last

polishing step um and the way that it works is that instead of things sticking to the beads

the things actually go your proteins go through and around the beads so these beads are filled with these little pores

these little like secret tunnels and there's different sized ones and the small proteins well they're going to get

kind of tied up going through all of those little tunnels but the big ones are not even going to fit in those

tunnels so they don't they get to take a shortcut you can think of it kind of like

um a series of roads and you have little cars and you have big trucks and the big trucks can't go through all of

those tunnels so they get to take shortcuts around them but the little guys have to go through all those

tunnels and so they're going to take longer to go through the column and therefore the bigger things are

going to come out first the smaller things are going to come out later there are a ton of different types of

columns that we can use um and they can be filled with different types of resin they're often like sugar

based or some sort of agarose or um there's some sort of dextrose there's various ones

stabilized stabilized sugars and so you might see things like super decks you might think see things like super

Rose I don't know how you spell that might be an O um but anyway you don't need to know

these different names but if you see something come up like that that's probably what it's talking about on this

size exclusion chromatography whenever you see a word in a method that you don't know what is just Google it

um and so if you Google it and it'll tell you it's a disclusion column and then you're like oh I know what size

exclusion columns are um without having to know like oh what's super Rose

um so Google is your friend more to when we're reading papers when we're dealing with methods um that's where you're

often going to go in order to find the basic information about like what the brand name you're looking at is actually

referring to um but then I want you to know basically the idea with size of solution

chromatography is that you're going to be separating proteins based on their size you might see this in preparative

which is what we've been talking about here which is when you're actually purifying a protein

um people also use it for like analytical purposes where you use a smaller column and you're trying to see

if proteins are sticking to one another because if they stick to one another they're going to be bigger and so they

travel together they're going to come out sooner um so I think we're going to look at

some examples of that as well later um but that would be your size exclusion chromatography

and that's often done as a polishing step for structural biology purposes so structural biology is the field of

biology that explores the interplay between biomolecular Form and Function so remember we've been talking lots and

lots about how the protein sequence is going to determine how the protein folds and that's going to determine how it

functions and oops this is one of my old figures where I have it the amino acid not drawn in its winter ionic form I

always wanted you to see I always want you guys to draw in your in this winter ionic form C even teachers mess up but

that should be in this weird or ionic form um because remember that non-ionic form

is really non-existent um so you should also you should have that negative charge on your Co minus

and the positive charge on your um Amino and terminal I mean immunogroup okay so basically structural biology

we're going to explore the connection between that form and that function and often what we're going to do is

we're going to look at this look at the form look at its structure um with techniques like extracrystallography

cryohe M NMR um and then we can kind of combine that with binding assays activity assays

various things to kind of test the function of these molecules and then we can make changes to the

protein or we can study changes that were naturally made like mutations that have happens maybe mutations that cause

disease and so that's going to be what you're going to be doing with your papers is looking at how mutations in

various proteins can cause these metabolic disorders um and often scientists will like want

to look at the structure want to do experiments in the lab kind of try to connect the two

and remember that because we can manipulate the sequence when we're doing that recombinant expression we can do

things like introduce those specific mutations or introduce new mutations um study domains in isolation all those

various things because of that recombinant technology that we can basically get cells to make any version

of a protein that we want so we talked about that stuff but now let's talk about how we actually go and

look at its structure so first off what do we mean by when we say like solve a structure basically

just means we want to like figure out how a molecules atoms are arranged in 3D

so remember our primary sequence that's just our sequence of amino acids and so basically we can draw a long chain we

can say okay well we got all these peptide bonds and stuff like this we know how which atom is connected to

which atom but that's not going to tell us how they're actually going to be folded up in 3D

and so we want to go and say okay well how are they folded up in 3D what is our secondary structure and our tertiary

structure and maybe even our quaternary structure we can use this using um using structural biology methods

so the main structural biology techniques we're talking about we're talking about extracrystallography

um this has kind of been the conventional Workhorse of structural biology lately there's been a revolution

in cryogenic electron microscopy or cryo-em um it's been like this revolution

there's been a bunch of advancements in the technology originally it was only good for like really big things but

they're starting to get down where it's also being able to be used for smaller protein structures as well as structures

and complexes and things like this um so we'll go into a little more in detail but still just at the very um

lowest level there's lots lots more detail but basically we'll take a look at these different techniques but first

there's also a couple more one is nuclear magnetic resonance or NMR

abbreviated NMR um which is good for small little little small flexible things and then there's

going to be kind of computational methods so you're going to see things like Alpha

fold Rosetta fold Etc and so we're talking about like AI

machine learning all this various things um there's also like molecular Dynamics

and things like this so lots of different techniques that you can do on the computational side

um but then these you have these which are going to be um

experimentally determined and so that's going to be like actual wet work

okay so those two main ones that you'll see these days are going to be X-ray crystallography and single particle cryo

em so we're distinguishing single particle um when we talk about a particle

basically that's just like that could be your protein that could be a protein complex it's the thing that you want to

you're trying to get a look at um and so there's also methods like um for for

electron microscopy that are not single particle that are basically looking at like tomography or basically like slices

of cells um things like that there's some really cool stuff going on in those fields but

we're going to be focusing mostly on the single particle the basic idea is that with x-ray

crystallography what you're doing is you take a protein and you get it to kind of crystallize and this is a lot harder

than it sounds and basically um what it means though is that all the protein copies are going to kind of line

themselves up in an orderly array so if you think about like assault and assault lattice how everything's kind of

interconnected in the same orientation throughout you get the same thing happening but with a protein

then what you're going to do is you're going to shoot X-rays at it and those x-rays are going to interact with the

electrons in the in the protein or whatever other molecule you're working with and then those interactive waves

they're kind of going to get scattered those scattered waves are going to interfere with one another and think of

dropping a golf ball into a swimming pool and all those like little Ripple waves um how they interact with one

another the same thing is happening here but we're dealing with x-rays and then they're going to go hit a detector when

they hit a detector they're going to get a pattern of make a pattern of spots called the diffraction pattern and then

scientists can work backwards from this pattern in order to figure out the structure of the molecule

so I did some of this in my grad school days um and it can be really cool but it can

also be really frustrating when your protein doesn't want to crystallize for you so you have to try out lots and lots

of different lots and lots of different conditions what about cryo em well with cryo-em

here basically you're kind of it's more like taking pictures but you really really fuzzy ones and so you can't just

take a picture of a single molecule that wouldn't be very helpful it'd just be like a fuzzy fuzzy thing so instead what

you're going to do is you're going to take lots and lots and lots of pictures of the molecule

and what's nice about cryo-em is you don't need to get it to um like crystallize you are going to freeze it

in place but it's not you're not making it kind of freeze you're basically freezing it by by literally freezing it

or at least vitrifying it so basically you get it super super cooled in a really thin layer of um thin layer of

water that serves as like this glass and then what's going to happen is that your proteins are kind of going to get

stuck in place wherever they are so they're going to be moving around randomly and they're going to be kind of

like so you might see some like laying down or standing up or things like this and

then what's going to happen is that they're just going to kind of get stuck in the position that they were in rather

than crystallography where they get stuck in that position that's going to be the same throughout the crystal

now what's going to happen is you're going to take lots and lots of pictures of these using electron beams and so

because they're so tiny we're going to be using um electron waves instead of light waves because light waves aren't

going to be small enough um so we're going to use these electron light waves and we're going to focus them um by

magnify electromagnetic lenses and then they're going to hit a detector and then there's a bunch of computational stuff

that will give us those fuzzy pictures we get fuzzy pictures of the protein and all sorts of different orientations and

we kind of average them together so we'll say okay well those ones all look like they're laying down and these ones

all look like they're standing up and so let's put these together and then okay well now we have the same thing in

different positions so we can average those all together we can use them to make a 3D model similarly to um say if

you were doing an MRI or something like this um where they take different slices and

then they kind of like work backwards two piece the 3D thing together and then you're able to figure out what

the structure is based on averaging together all those fuzzy images you can also with cryo-em because

they're kind of can be indifferent things can be in different conformations as well and sometimes you can

um see those too so for example you might see someone sitting instead of standing or laying and when they're

sitting well then they're in a different shape and so if you can isolate like okay well we have ones that are sitting

and we'll isolate those um and then you have ones where you can see the back of the person sitting once

you can see the front of the person sitting we can average all of those together so basically with cryo em

there's ways to see different orientations of molecule like different conformations of molecules as well as

just being able to average together all of those different um kind of like orientations of the same

of the same confirmation bottom line you need a lot a lot a lot of data um you average them all together

and you can get a structure but it's not as easy as it sounds and with any of these techniques when we're

talking about cry OEM and we're talking about crystallography what we're getting is actually not we don't get the

positions of the atoms directly instead we just get the evidence that they were there

so when we're dealing with crystallography we're dealing with kind of like evidence of where the electrons

were um and so when we're dealing with um when we're dealing with prior again

um then we're dealing with like technically we're dealing with like coulomb potential

um anyway you get this like fuzzy looking kind of map thing and then the scientists have to go and kind of model

things into those Maps so when we go and we look at structures what we're looking at is actually a

structural model where they're modeling in the positions of the atoms into that data and so we'll look at how we can

actually go and explore these models ourselves the easier the higher the resolution of

the data kind of like the less fuzzy it is and the crisper the um the crisper the maps are going to be the easier it

is to fit in the model so we talk about resolution a high resolution is going to have little numbers

so high resolution Little Numbers

low resolution pi numbers high resolution is going to be good low

resolution is going to be bad which you can probably um guess based on if you think about a

high-res TV versus a low risk TV you want that high res right well resolution is referring to how close two things can

be well you can still kind of like tell the two different things and so the smaller the number the um the closer

things can be where you can still tell them apart so it's like if you have two dots far away can you tell that there

are two dots or do they just look like one dots um and basically the better your

eyesight the closer those dots can be and you can still see that they're two different things so similarly the higher

the resolution of our data the more easy the easier we can kind of say okay well yes this is there's an atom here there's

an atom here there's an atom here versus oh there's some sort of sausagey thing will have to fit things into

um so that's what you'll see when we talk about resolution and we're typically dealing in terms of angstrom

which remember is a 0.1 of nanometers what's really great about structural biology for the outsider is that all of

the structures and the experimental data behind them like their maps and stuff have to be deposited so that anybody can

go and look at them and they get deposited into this thing called the protein data bank or the PVB

you can find all sorts of information about how the scientists solved it you can actually go and explore the

structure you can check out different features about it and so we'll be looking at the pdb and they have some

great educational resources so check out um pdb 101 or more for more info on it on how to

use the pdb as well as more info on structural biology another technique is nuclear magnetic

resonance so this is going to be good for small flexible

what happens with structural with um crystallography and with cryo-em is that basically there can be regions of the

protein that don't resolve so you don't actually get maps for those regions the part of the protein is actually there

but the data is too fuzzy so you can kind of think of it all averaging out um it's all blurring each other out

canceling each other out and so you're not going to see those regions that are flexible

but with uh with NMR here it can actually use those flexible regions but it only can do deal with small things

um but they're going to be like randomly moving around and what you actually get is some sort of Ensemble and so you can

check out the video if you want to learn more about this the PBS and A little video of explaining it

um but we don't need to worry about NMR for the purposes of our class um the other thing you'll hear a lot

about is like these Ai and machine learning various ways to predict structures and you'll actually see that

in uniprot so it's going to be the protein database we'll look at um basically there you can actually see

predicted structures as well um so there are limitations like if you want to look at complexes if you want to

look at regions of proteins that don't have structures that are like known structures kind of or regions that are

kind of um more flexible if you want to look on modifications there's various

limitations to these prediction mod algorithms but they often can be helpful for structural biologists to kind of

like get an idea of what a protein looks like and maybe even use it to help make their models so there's various things

like this um that you'll probably hear about Lots so that's the basic idea we can

basically get cells to make a protein for us using recombinant expression techniques and we can break those cells

open and then we can take a mixture that was from the broken open cells and we can use protein chromatography to

separate the proteins in that mixture based on how they do or don't interact with various resin um so these little

beads that we have in these columns then scientists can use structural biology techniques if they want to go

and take a look at it they can also use um biochemical assays and things like this to study the functions of the

proteins that they purify