Shadowing Practice: [TALK 13] Structure Prediction and Design using AlphaFold – Sami Chaaban - Learn English Speaking with Video

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Alright, yeah, so let's talk about AlphaFold.

I'll be talking about structured prediction using AlphaFold, and then at the end, I'm going to talk about design.

I'm going to give more of a higher level kind of view of AlphaFold

and give more of like a user -centric focus and kind of as a user and at the LMB,

you know, how to use it and what you need to know in order to properly interpret the results.

So, yeah, this is the breakdown.

We'll start with the overview, talk about how to write predictions, especially at the LMB.

I'll talk about some tips and then some things that you can use alphabetical prediction for.

And at the end, we'll talk about designing your own protein binders.

All right.

Well, first of all, why would you need structural prediction?

Well, you might just want to know what your protein looks like, right?

I mean, it's very important to give this structural context to your biological questions, right?

But really, I mean, a lot of the applications have been to generate models that help interpret structural data like cryo -EM, crystallography,

and other structural data that either you want to use as a starting point

or you want to help interpret lower resolution features.

You might want to use it for protein engineering for designing constructs.

How do I cut my proteins so that I can purify better?

And we'll talk about that later.

But really what you want to do is quickly generate these hypotheses

that can be tested by these other techniques that we've been talking about.

And this progress towards predicting a structure purely from its sequence has been monitored by this competition called CASP.

And, you know, over the years, the developments in neural networks and how, you know, proteins can be predicted.

And, you know, around 2018 is when AlphaFold came out, which did really well.

And then 2020, you know, AlphaFold2 came out and it did extremely well.

And, you know, anecdotally, 90 % is where you would consider comparable to experiment different.

In any case, these are the results that they were showing.

You see these targets.

So these are proteins that had not been seen before by these algorithms, but then were predicted with quite good confidence.

So you're seeing the global distance test here, just an overall measure of how close you are to the actual structure.

And so then over the years, we've now come out with alpha -fold multimer and alpha -fold 3.

And kind of alpha -fold is an aggregate term for these things, right?

So we have the ability to predict multimeric proteins or now with other molecules like DNA and RNA.

Okay, so how does it work?

The insight comes from available structures on the PDB.

So this is a database online where experimentalists will submit their structures.

And now we have a sequence to structure relationship.

And there's many on the PDB.

And this is what can be used by these neural networks to train a model

that will be able to predict the structure of a new sequence it hasn't seen before.

But one of the insights that was really important, which had been also already in development over the years, is insight from a multiple sequence alignment.

So you look at your sequence of interest, but you also look at the related sequences.

You can see there are residues that are evolving in complementary ways.

And so that's a hint that these might be close to each other in space.

And so this co -evolution is a very important signal in these structure predictors.

And in a way, you can think about the multiple sequence alignment as actually being the input rather than just the sequence.

So that's what we're what I'm calling the multiple sequence alignment structure predictors.

So we have alpha fold, which I'll be mainly talking about.

There's also Rosetta fold from the Institute of Protein Design from David Baker, which is also very accurate.

And again, these you're generating a sequence alignment and you're getting out structure.

You should be aware that there's also these protein language model structure predictors.

This is like ESM fold or mega fold where it doesn't actually generate multiple sequence alignment, but rather directly generates a structure.

And you can think about it as the evolutionary information has essentially been embedded in the model.

So it may suffer from the same things, but it runs a lot faster because you don't have to generate sequence alignment.

All right.

So how does that fold work?

Just a high level view.

I'm not going to get too deep into it.

But like I said, we have to generate a multiple sequence alignment.

And the way to do that is to search, you know, database.

So we have things like your F90, Magnify, or BFD.

These are either metagenomic databases or some clustered uniprot databases

and from those we can extract related sequences to our sequence of interest, generate multiple sequence alignment that then goes into this first model,

the first part of the algorithm, which is the MSA module.

And by extracting those relationships, we can now just generate an initial estimate of where these XY coordinates of the different residues should be.

Another thing that can be input into this module is, you know, examples of structures that are similar to your structure right

so these are templates it turns out that these are not as important

but you should know that they're also something that helpful can fetch from the pdb let's say and insert into

this MSA module.

Now, after we get our initial X, Y, Z coordinates for residues, then it goes through some other modules.

So this is a structural module that then gives you something that looks like a protein.

And for AlphaFold2, this was a structural module, but then for AlphaFold3, this is now the diffusion module.

So instead of treating the residues as proper geometries, it's basically a denoising step that's turning it into something that looks like a protein.

And because of this difference, actually, you end up with sometimes quite different results between AlphaFold2 and AlphaFold3.

And some of the things, you know, the caveats of FF3 might be coming from this difference as well, which we'll talk about later.

But essentially, once you get that, and it's not accurate at first, so it goes through this recycling step,

so it gets put back in for a few recycles until it basically converges on something

that is as confident as it can get.

All right.

And you can slap me at any point, by the way, if you have any questions.

really all you have to worry about.

Oh, I should talk about ColabFold.

Yes, so this is the AlphaFold framework in general.

Now, you probably have heard of ColabFold.

And essentially, ColabFold is an implementation of AlphaFold that is on the Google Colab notebook, which is why it's called ColabFold.

But also, it has a multiple sequence alignment search that uses MMCs,

which performs a clustering step and also a pre -filtering step.

In any case, it's a faster version of obtaining

that multiple sequence alignment alignment and that's what we have implemented at the LMB locally.

So when you hear call AlphaFold, it's essentially AlphaFold with the MMSeq database search.

But really all you need to worry about as a user is, you know, your sequence.

That's what's nice about these things.

You give it your sequence and you get your structure.

Now with AlphaFold you'll get five structures because they train five different models in slightly different ways.

And so these are five different attempts at getting the right answer

and then it will rank them and so hopefully one of them will be correct.

You'll get your multiple sequence alignment.

So this is what was generated in the pipeline.

Here's an example from this run.

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I'll talk about it in a second.

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And then really importantly, you'll get these confidence metrics.

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For example, we get global metrics like the predicted template modeling score or the interface predicted template modeling score.

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This is basically one number to say how accurate we feel this structure is and where higher is better.

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But then you also have local confidence metrics like PLDDT or PAE, which we'll get into.

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And these are, you know, what is my confidence about the secondary structure or about two residues interacting?

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So I think these are really important.

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So we'll talk about them in detail.

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And something that I just want to highlight here is that, you know, for things like PTM and IPLDT,

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these are numbers that are better when they're higher.

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The PAE is an error.

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So a lower number is better, right?

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Lower error is better.

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I see people make this mistake quite a bit.

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But yeah, we'll get into those.

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So here is an example of a protein that I work on.

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So I work on a motor protein, dynein, and this is a dynein -dynactin complex.

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And just part of dynein is this, you know, subunit called the light intermediate chain.

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It's a good example because it has this globular domain.

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It has some alpha helices on either end.

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And from which you predict just this protein on its own, you see you get five structures from alpha fold.

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You can see these alpha helices are kind of floating in space around the globular domain.

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You know, one shouldn't necessarily take those positions of those alpha helices seriously.

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If they're, you know, stuck on a string, it just kind of plays randomly.

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But these five structures are ranked by the algorithm.

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And over the years, different ranking metrics have been used.

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For example, average PLDDT or just that PTM number or the IPTM number or some combination of these things,

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80 % of this, 20 % of that.

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so in general what i recommend is that you actually look at all the five of those

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predictions and i'll talk about some special cases where that's important

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but yeah i mean typically users will just look at the top ranked one

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and it might not be able to give them the answer

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that they want the other thing that's generated like i said is the multiple sequence alignment

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so here you're seeing all of the sequence

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that were pulled out of those databases across the position of the protein and here's the concept of msa depth right

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So the more sequences in the multiple sequence alignment, the more the depth, so the more robust the prediction is going to be in that region.

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You can see those regions in the NNC termini that have less depth, so they may not be predicted as robustly.

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So that's good to look at.

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I mean, typically you would want more than 30 sequences, although you can see here, you'll get thousands in many cases.

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Now, the first important metric is this PLDDT.

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So this is a local confidence metric

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so it's you know c alpha distances within 15 angstroms of the residue

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and so this is a really good way of figuring out

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if you have confident secondary structure so one rule of thumb is below 50

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and it's probably disordered so there's no secondary structure

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if it's above 50 then probably the secondary structure is created with confidence so in this example.

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You have this region of the protein that is disordered.

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You can see the spaghetti is colored in red, and those are the low PLDDT regions, whereas these alpha helices are high PLDDT.

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So if you're asking questions about secondary structure, it's better to look at the PLDDT.

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And I'm just showing what AlphaFo's recommendations are for what these numbers mean above.

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So yeah, 0 to 50 is disordered for bad prediction.

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Now, if you're interested in how residues are interacting across a protein, then you want to look at the predicted aligned error.

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So here's each one of the five ranked predictions with their PAE plus below.

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And so let's just focus on the top ranked one here.

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So what you're asking with the PAE is what's the expected error of residue one

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if you align the predicted and true structure at residue two,

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which is a bit confusing because we don't actually have the true structure in this case.

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So that was the power of Alpha Fold is

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that they were able to train this metric based on when they did have the true structure.

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And basically, how does that confidence turn into an error?

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And then can we apply this to things that we haven't seen?

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It turns out to do quite well.

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And let's talk about this plot a bit more specifics.

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So if you look at this part of the plot, we have a question of what is the confidence of residue 198 versus itself?

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Of course, it's very high, which is why you end up with this diagonal across the PAE.

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A more interesting question is, for example, what's the confidence of 198 versus 113?

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It's actually not that interesting, but it's a good example of where you have very high error.

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So the confidence of the position of these two is actually not very good, which makes sense.

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I mean, they're very far apart.

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Now, it gets more interesting when you're asking things within a domain.

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For example, here I have two residues

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that are close to each other whether i'm asking 324 versus 256

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or vice versa you can see that we're getting a very low error

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so it's very confident in the relative position of these two residues

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so we can you know make a hypothesis

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that these two may be in a domain or interacting with each other or something like this

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and so what's interesting is that

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when you have these domains then you start to become confident in the position of your neighbor

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and your neighbor's neighbor and your neighbor's neighbor's neighbor

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and then you get this extension away from the diagonal of this low error region

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so that's when you get domains so you get these like square um regions or you know extended regions

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if we keep going down you see this loop there's not much to say within the pae

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and we have our next sub domain and

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when you have two sub domains that are interacting with each other

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that are far away in sequence space you end up with these blocks

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that are away from the diagonal right

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so here we have this region of the protein is interacting with

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that region of the protein

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so you get you end up with these regions of low error that are outside of the deck

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for things like secondary structures it's not really that informative you can see

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that the extension away from the diagonal is actually not

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that large because the alpha helices are quite small and again

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if you're asking questions about secondary structure you'll be wanting to look at the plddt rather than the pae

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so what about protein protein interactions it's the same logic

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so here we have an example of this subunit

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that i showed you before and this other part of the dining tail and you know

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if you give any implementation of alpha fold more than one

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chain essentially what's going to happen is it's going to use alpha fold multimer

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so you usually don't have to worry about

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which model is going to be used is alpha fold is it multimer typically it's already handled for you.

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And for this protein -protein interaction question, we have these different PAEs for each.

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We look at one.

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It's essentially the same thing, right?

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So we have protein A with itself.

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This is identical to what I showed before.

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It has its diagonal.

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It has extensions from the diagonal.

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So these are subdomains interacting with each other.

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Same thing for B.

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But what we're really interested in are these quadrants of protein A interacting with B or B interacting with A.

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so that's what you're getting out here right

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so we have regions of low error um between the two different proteins

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which is the same logic now you might already be asking yourself why is it

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that when we're asking a versus b

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and b versus a are we getting slightly different results like why isn't this perfectly a perfectly symmetric plot

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and here's a dramatic example of this where we have this little peptide

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and this um you know globular domain and you can see you know if we're asking B versus A, we're getting a lot of error,

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but if we're asking A versus B, we're getting less error.

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And so what's happening here?

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That's really not clear what's going on.

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I mean, these things are generally black boxes.

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But the way that I've understood this is that

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if you have one protein that has a lot of variability or low confidence in its position,

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whereas another one has a lot of confidence in its position,

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like a bigger domain, then sometimes if you ask a question of one versus the other, it'll be quite high error because it's moving a lot.

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Whereas Yes.

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The big one versus the small one, you'll get less error.

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So what I would do as a user is just treat this as a red flag.

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Maybe this prediction should be taken with a grain of salt.

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Right.

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And of course, all of these things need to be tested by other techniques,

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but I should I should flag this because I get this question a lot.

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And so this is I mean, that this is probably the most important part of the whole talks.

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If there are any questions, I'm happy to take them now on the PAE. Or the.

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I'm going to do that.

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Good.

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Now, sometimes it's hard to kind of read these plots.

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So I find things like ChimeraX where you can highlight certain regions of PAE really useful

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and then it'll map where that is on the structure.

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So this is just implemented in ChimeraX.

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There's also this PAE viewer web app where you can load your structure

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and you can also load cross -linking mass spec data onto this, which is really nice.

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You can see which are satisfied enough.

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All right, so those are the output.

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We have a structure is a multiple sequence alignment and our metrics, and depending what kind of claims you're making for your project,

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you'll want to submit different things for manuscript.

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If you're making interpreting the interaction between different proteins and then continuing further in your project based on those,

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then you'll want to present the plot for this prediction.

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You're making claims about secondary structure want PLDDT we don't usually submit things like you know the multiple sequence alignments

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or the other

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ranked predictions but yeah please submit your PAEs

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if you're talking about protein protein interactions in your manuscripts it's also nice to actually deposit these predictions

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so you know people can actually go

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and see what they look like it's not really appropriate to submit them to the PDB

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because those are experimentally derived structures and so something like mono archive

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which is a nice place that you can deposit these things

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so here's just a recent example from kelly's lab where they deposited this nice structure from alpha fold 3 prediction

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and um you know so that other people can check it out all right

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so how do you actually run your own predictions actually the simplest way is to just fetch a prediction right

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so alpha fold has run essentially all human proteins through its algorithms

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and submitted it into this database or I've released it into this database, but it's also integrated into the Uniprod.

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So if you just go in the structure section of Uniprod, you can see the alpha fold, what your alpha, the structure is.

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Now, like for something like, you know, RL3 here, this globular protein, you see this one looks like in Uniprod,

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you know, it makes sense to just fetch it from there or look at it on the website.

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But for things that are, you know, dimers or, you know, multi -protein assemblies, you can end up with something like this,

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where it's just like an extended alpha helix whereas we know that this is a coiled, coiled protein.

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So if you run, you know, a prediction yourself, you know, you'll get the actual structure of this cold coil.

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And so yeah, typically you'll want to run your own structures,

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the prediction sorry, and so a simple way to do that is just to go on the Google Colab notebook.

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Remember I mentioned Colab fold.

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This is a Google Colab notebook,

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so hosted by Google where you can put your sequence and have it run on Google's GPUs and then you know, obtain the prediction.

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um and you know if you just visit um

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that website then you'll see all the different options it's not just colapful

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that they have the problem with this is

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that you have to kind of stay on the page

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and the tip of the page might time out and

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if you know

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if you want to use their nice gpus

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you have to pay a little bit of money now what's nice about the lmd is

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that we have a local installation of colapful and

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if you're not familiar with scientific computing then um i encourage

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you to visit i mean you should visit the scientific computing uh website read their guidelines on how to use the cluster,

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how to submit jobs, and your practices.

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But essentially, once you have all of that in place, you just need to add these lines to your .cshrc or .vashrc,

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and then you'll have access to the colapfold command, where you can just provide it a FASTA file with the sequences that you want.

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Just run colapfold to that FASTA file.

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You'll submit it to the cluster, do its thing, and then sometime later, you'll get your predictions in that directory.

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And there's a lot of options which you can check out

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if you just run Quolap Fold on its own to see what your options are for submission.

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Some of the things that you can play with, for example, is playing with the number of recycles.

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I mentioned that recycling is getting more attempts at getting the right structure.

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This is usually handled internally where it reaches some tolerance or if it gets to 20 recycles, usually you don't mess with that.

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You can also provide your own template.

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So maybe you have something, a starting point for AlkaFold and you can provide it as a PDB.

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This hasn't worked really that well for me, but for some people it has worked.

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So that's an option that you should know about.

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I didn't show this, but this is just an example of recycles where you can see that the

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prediction kind of plateaus at around 10 to 11 recycles.

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And so in this case, that was 11 was sufficient, but usually you just kind of let it handle it internally.

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Something interesting you can do is actually sample confirmations or try to sample confirmations.

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Alpha -folds will sometimes just predict the open state of a protein or the closed state.

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Sometimes you want to get it out of that and try to predict other confirmations.

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And something you can do is just by introducing uncertainty into the model.

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So like I said, the deeper the MSA, then the more robust the prediction.

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But if you want to actually introduce uncertainty and get it out of some local minimum,

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then you can just reduce the number of sequences in the multiple sequence alignment.

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And then hopefully that uncertainty will lead it to one another structure getting that other confirmation.

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You can also increase the number of seeds, which is basically a random initialization of the model.

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And if you have different seeds, like two seeds, then you'll get two per model array

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so you get 10 structures in the end and then you can start sampling a lot of different um

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starting points or you can just randomly switch off parts of the network apparently

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that works as well for some people here's an example with many seeds with this dropout option

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you can see this protein has kind of sampled these different conformations

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so it does sometime work

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and you know you just have to try these things right um an important one

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if you're doing like prior oem model map building models into maps let's say

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you might want to use amber relaxation

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which is you know helping the sidechains get into the proper geometry

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because you know alpha fold might have things that clash aren't quite correct

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and so amber will help them kind of relax into a place

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that is a good starting point for for model building all right

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so that's alpha fold two alpha fold three

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there's this really nice online server from deep mind where you can just put your sequence in

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and and let it run on their servers.

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You just need a Google account to be able to do this.

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The only caveat here is that you only have 30 jobs that you can do per day.

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But it's really nice because you can essentially add different types of molecules.

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You can add DNA, RNA, ligands, ions.

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Here's, for example, an ADP molecule that I've added to this protein.

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If you're interested in different types of molecules for structure prediction, you should also check out RositaFoldOatom.

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This one has like this nice web interface that you can use.

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And then you get this prediction out.

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This is a really large protein that AlphaFloat3 web server lets you run.

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And you can see the PAE plot.

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You can see what we talked about here, the PLDDT colored, the IPTM and the PTM scores, all the things that we talked about.

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And you can download this, the five structures that it's predicted.

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The only thing that you can really play with here on the online thing is the seed, right? of the different random initialization of the model.

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So you can try different seeds if you wanted to see if you could get different results.

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So let's talk about the difference between the two.

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I mean, at a higher level.

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So what's nice about AlphaFull 3, of course, is that it handles different types of molecules, like I mentioned, whereas AlphaFull 2 doesn't.

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It's extremely fast.

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They've sped up the MSA portion, and the diffusion module that I mentioned is also quite fast.

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and this web server of course is running on google's um server

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so they're also very fast and

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so you can run up to 5000 amino acids as well um

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which is really nice of course it has limits you can only run up to 30 jobs

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and it can produce a few more hallucinations so especially in regions

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that are disordered it tends to make them ordered

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they give you alpha helices where there should be i know a lot of people have noticed this

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so alpha full 2 is also you know still very nice to run I don't think it's necessarily been replaced,

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but of course it only handles proteins.

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But yeah, it can be more accurate for multiverse.

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Here's just some anecdotes about alpha -3.

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This person is saying it's the end of the world as we know it.

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After this transcription factor has been correctly predicted to bind to this DNA.

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But then again, you have people that say, you know, it's not able to do predictions properly,

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but rather has predicted a ballet 2 -2 structure for this routine complex.

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So I think you just have to try different things.

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Something I want to mention also is another injection of information into AlphaFold, which is in this software called AlphaLink,

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which is essentially taking cross -linking mass spec data and introducing that into the pipeline.

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So if you think about cross -linking mass spec data, you're essentially getting information about the relationship between residues right the positional relationship

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and so if you just put

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that in as weights to the msa module you're essentially improving what it's already picking up from the msas

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so here's an example for

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this protein complex where alpha fold has not predicted these in proximity

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yet with alpha link due to the cross links that were predict that were experimentally measured

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we can uh alpha can more correctly predict this structure

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so we have this implemented here

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if you just do collab link 2 um your fasta

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but now you also give it your cross links um you can also just run this and uh

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if you just run collab link 2 without arguments you can see what the um what the options are for

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that all right so i got some tips so like i mentioned

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if you have really big proteins

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you still have to start thinking about what you're submitting to

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a full three server like i mentioned has up to 5 000 residues the notebook

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you can get to one to two thousand

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um on so the gpus on our cluster you can get you know two

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and a half three and a half thousand depending on

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which cluster node you submit to but i mean even for the some things

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that i studied like the dynein heavy chain is four and a half thousand residues, right?

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And so you could try getting, you know, very close to the maximum limit, but then something like alpha 3 will,

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you know, predict this shrimp -like structure for dynein, which we know is not correct.

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And so typically what you want to do is segment out your big proteins into smaller portions, smaller domains, smaller regions of interest,

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and then maybe stitch them together if that's your goal.

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So this is what I've done for this large protein.

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I mean, sometimes it can even help with accuracy.

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You can see here this cold protein that has this spaghetti structure between these two cold coils.

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But if you segment it out and then do predictions of different segments,

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you can correctly get the intermediate cold coils that we know are there from other techniques.

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Something to watch out for.

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I mean, false negatives are always going to be a thing, right?

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but here's an example of this little peptide binding to this long -closed coil

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that we have experimental evidence for that is not predicted.

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I wanted to mention this

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because in this case we just truncated the protein a little bit more

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and all of a sudden it's able to predict this interaction.

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So sometimes you get some weird phenotypes like this that you want to play around with, you know, where you're truncating things.

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Much more scary are the false positives.

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Here's an example where I've taken this protein in green and I've just chopped it in half.

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So basically I'm exposing what would be varying residues.

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And I have another protein that's basically not related.

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And you can see that it's basically taking this beta strand

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and appended it to the core of this protein with a very nice PAE.

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So we're interpreting here, right, this PAE plot, very nice in these quadrants, whereas this is impossible.

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So you have to be really careful about these things, you know, where you're chopping things.

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And of course, all of these things need to be further verified by other methods, like the ones that we've been talking about.

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I didn't mention that you can get model -to -model variability, right?

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You get five predictions.

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So here, four of these predictions are showing that this peptide is interacting with the cold coil here.

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But then if you look at the fifth one, we're getting a different part of the peptide interacting with a different part of this cold coil.

412

And there's some NMR data suggesting that this peptide in its entirety can interact

413

and so maybe this is something that warrants further investigation right

414

so this is something that came out not just from the first ranked result

415

but also from the other results

416

or you get cases like this where you know one of

417

them will be open close well some of them will be closed closed

418

so just something you got to watch out for when you're

419

when you're running these things all right let's talk about some applications

420

so i think the simplest to understand is when you're doing structural biology

421

and you have lower resolution data you want a complementary method to see

422

if you can interpret the interactions i think alpha fold is great for this here's something

423

that clinton um put together for one of the structures he was working on back in 2015 of dining

424

so this is again part of this complex we have this big dynamic structure it's like a filament -like structure

425

and they were had a hard time fitting this p150 subunit into this density

426

And so what he did is he chopped it into different pieces.

427

He predicted the structure of each of these pieces, and then he could very nicely place them into this lower resolution map.

428

Now he has something he can work with, because now he can make mutations and test if this is actually where it docs using other methods.

429

Of course, it also helps with higher resolution structures.

430

I mean, here is, for example, a part of a high resolution structure that's not well predicted or not well resolved, like this little loop.

431

And, you know, if you just predict a structure with Amber Relax on, maybe it will help you get the starting point for this little loop.

432

And then you can maybe work with that and see if there's something you can build into it.

433

I didn't mention protein engineering early on.

434

Here's that cold coil that I mentioned.

435

And here I was trying to purify this protein and accidentally truncated it at this four helical bundle.

436

And it turned out that based on alpha -folk prediction,

437

I had cut right into this region that would actually be exposing part of the cold coil core.

438

And so then I just changed the truncation to this other region, and all of a sudden it became soluble.

439

So it doesn't mean that's why it worked, but just something it's nice to actually see where you're truncating your proteins when you're making these designs.

440

All right.

441

Something else you can do is screen for interactions if you have a database of,

442

let's say, So you have a cross -linking mass spec data set, or you have interaction screens deposited online from the BioGrid.

443

There's something like AlphaPullDown that lets you then do alphaful predictions of everything

444

and see if you can get new predictions out or something that you can run further experiments on.

445

I've written something called AlphaScreen, which just takes things from the unit prod and it makes little FASTA files for you to submit to our cluster.

446

Admittedly, this is a bad name given the AlphaScreen that was mentioned this morning, which is an actual essay.

447

Anyway, when you're doing these things,

448

then it starts to become more important how you rank these proteins because you might have hundreds or thousands of predictions then.

449

And, you know, there's things like the IPTM.

450

There's a PAE in that quadrant.

451

Is that a good thing to look at?

452

There's this local interaction score, which is some aggregate thing.

453

Then you have these docu.

454

I think the way to think about this is that by ranking, you're essentially just shortlisting these things.

455

And then really you need to use your experience

456

and carefully look at each structure to actually see which ones are worth further investigation.

457

Something else to think about with structure prediction is the ability to look at structural homology.

458

So we have, now if you have a domain

459

and you want to see all of the things that look like this domain but don't necessarily share the same sequence,

460

they're not homologous, you can use something like progress, which comes from the LMB, from Joe Greener, and Bold Mason and the Valley,

461

for example, where you can run these things through an alpha -fold database,

462

and maybe there's something that hasn't been solved that you can then further investigate.

463

Here's an example of a hook domain where you can see the alpha -fold database has been used to extract structural homologs.

464

So that's something that might be interesting to you.

465

I'll end this section with just some limitations and some advanced use cases.

466

I mean, it's important to think about isoforms when you're thinking about structure prediction.

467

You might have an isoform that is predicted to bind, and then another isoform that you don't think should bind, but is still predicted to bind.

468

Because the way that these things work is that you have an interaction interface.

469

If there's a lot of homology at the interface, then small changes between isoforms that should break this interaction actually might not be picked up.

470

And so you might get false positives from isoforms in the family, which we've seen.

471

Well, the numerization state isn't always obvious, right?

472

So this protein, is it a cold coil or is it a three -way cold coil?

473

It's not clear, right?

474

There's not an obvious metric you can use to say, actually, this should be a dimer, not a trimer.

475

We talked about alternate conformations and playing with the MSA.

476

There's also a way you can cluster the MSA, right?

477

You can take sequences that look like each other and then put those and use those as input,

478

and then take other sequences that look like each other

479

and use those and maybe those relate to different conformations of the protein right

480

so you can play around with the msa as a way of getting those out

481

and that's something like an alpha cluster let's say

482

of course post -translution modifications aren't going to be there right

483

there's not going to be a phosphorylation from alpha fold

484

so sort of might be very important mutations may not break

485

an interaction for the same reasons the isoforms won't right

486

because one residue will not change the a co -evolutionary signal of that interface necessarily.

487

So I've tried this with computations that I know should break the interaction where alpha fold will still predict interaction.

488

So generally, one should not use alpha fold to predict if mutation will break an interaction.

489

There's some evidence that rosetta fold might do better for this, but I mean, you should be careful with these things.

490

Ligens and nucleic acids are now handled really well in alpha fold three

491

and rosetta fold all atoms so that's not something

492

that we have to worry about anymore now whether

493

or not these things are accurate we're still you know it's still early

494

but it seems like it's working for many things

495

antibodies are difficult

496

because you have these rapidly evolving regions of protein of an antibody

497

that's trying to you know bind to its antigen and

498

so these rapidly evolving bits aren't really well handled by things like alpha fold

499

that use you know co -evolutionary information over long you know, over a long historical time, essentially.

500

And so there are dedicated things that you can try to run, but this tends to be a limitation.

501

And for proteins with very low homology, which essentially means that they don't have a lot of related sequences,

502

so the MSA depth is going to be very low.

503

Those things also...

504

are limited by this method.

505

You can try things like curating your own multiple sequence alignment, which can help.

506

We're using these protein language models, for sure predictors, which don't need an MSA.

507

But they may suffer from the same caveat, since the evolutionary relationships are essentially embedded in them.

508

So it might not actually solve the problem, but worth trying.

509

All right.

510

Are there any questions on this part before I go to find your site?

511

Go ahead.

512

okay

513

yeah we work with a lot of clothes i have seen that before i don't know why

514

in some cases it does it doesn't in the wrong orientation

515

but yeah something to be careful about yeah no not really um you could you do

516

yeah no i don't have any i haven't i haven't had

517

that problem enough times to have to think about a trick for

518

that um you could try different lengths i mean that's the only thing i could think of right

519

if you give it a longer full coil to work with maybe it'll then properly place it but that's yeah anecdotal

520

anything else all right

521

so what comes out of the alpha fold era is this ability to hallucinate proteins now

522

and that's essentially what binder design is you're trying to design a protein

523

that binds another protein using the kind of algorithms

524

that have been developed for alpha fold

525

and this is the general pipeline right you have a target

526

you want to bind something to where those yellow residues are

527

so those are hot spots and you want a certain length of protein let's say 60 residues.

528

And then what these pipelines are doing is hallucinating a backbone.

529

So either running it through alpha fold and just hallucinating a protein, seeing if it binds, and then generating some loss function so

530

that it continues iterating until it actually figures something out that might actually fit that space or something like RF diffusion.

531

And then once you have a backbone, you can optimize the residues within it using something like protein MPNN.

532

And then that will give you something

533

that actually looks like a protein is soluble maybe you can express

534

and purify it and then scoring

535

that okay does this look like a protein protein interaction is

536

the very surface area good is our predicted energy of binding right I mean are the alpha fold metrics

537

you know reasonable

538

and then you can use your top rank ones to then test using my physical techniques

539

and I think now what's nice is

540

that a lot of the difficult parts of this have been package together

541

so that you can just run your target through something like

542

Minecraft or World Shin and then you just get your list out on the other side

543

and not have to worry about you know necessarily installing each one of the individual components and so for something like

544

Minecraft

545

which uses this alpha fold hallucination here

546

I'll just show you the figure from the paper right

547

we have our target protein again we're using alpha fold to hallucinate some backbone

548

we're using this mpnn to optimize the sequence

549

and then we're running through alpha fold again to say okay does this look like a protein

550

you know will it bind and what's nice is

551

that you know more recently the success rate of these binder designs have actually gone up quite a lot

552

so we have now nanomolar affinity binders in you know in these cases at least you're getting quite high

553

success right

554

so I mean in some some cases

555

it is you know like a hundred percent with this case of course

556

this it depends on your target some targets are very difficult

557

and very hard to design binders for which I've also experienced

558

but what's nice about this is at least a simple input you know there's minimal intervention involved

559

and depending on your target and you know the success rate might be quite good

560

and at the MLB you know you can run this quite easily

561

if you just run bindcraft

562

and then a json file you'll essentially run it on our cluster

563

and this just has you know where you want to run

564

it what it's called what the actual target is the pb file

565

which chains where on

566

that chain you want to actually target this binder how long

567

you want this binder to be the range of lengths

568

and the number of final designs before it actually ranks them

569

and so here's something to think about is how big is my target how big is my binder

570

You don't want to make it too big because then it's going to just take too long to get enough binders.

571

So you want to keep it less than 500, let's say.

572

And there's also other parameters you can play with.

573

If you run the Bindcraft just by itself, you can see where these advanced and filter parameters are if you want to play around with them.

574

We can talk about it if you want.

575

But essentially, you'll get, let's say, hundreds of designs.

576

Here are some examples of ones that I run, just a subset of them.

577

And, you know, some of the things that I've noticed is that there are,

578

you know, sticky parts of proteins that binders will like to stick to, especially with this Minecraft implementation.

579

You know, it will like to bind to a hydrophobic region of your protein, for example.

580

And so, you know, sometimes you're kind of stuck.

581

Sometimes you can play with different parameters.

582

Like if your hotspots are ignored, you can maybe mutate those non -hotspot regions to lysines and see if you can trick it.

583

Or you can try playing with the interchain contact weights and then, you know, see if that will work.

584

But essentially, you'll end up with this long list and, you know, you got to cross, you have to, you know,

585

purify these proteins across your fingers if they actually bind, right?

586

you can use additional metrics you know like alpha 3 predictions

587

if alpha 3 doesn't predict this interaction of the binder maybe

588

it's not worth investigating further for example um i've used

589

that in my pipeline you know recently i had you know

590

i ordered 20 binders out of a list of like 600

591

and about half of them bound

592

so this actually it seems to be working for some targets for others i can i could get any problems

593

but it's a nice tool so i you know encourage you to try

594

if That's something that might be useful for either alternating confirmation of a protein, blocking a site, and all the things that you might want,

595

labeling, for example, all the things you might want a binder for.

596

All righty.

597

That is what I wanted to talk about.

598

These are just some resources, some things that I found useful.

599

For example, how to interpret outfold structures from the MPL is very nice.

600

This is the original talk about Colabfold, where they naturally outline this MMSeq and the alpha pipeline.

601

The illustrated alpha fold is an excellent write -up of alpha fold 3 in great detail

602

and a comparison with alpha fold 2.

603

And then Rosetta Commons has this course online, this YouTube course, about structure prediction and machine learning approaches and structure

604

structural biology and I would encourage you to look at that if that's something you're interested in.

605

We also have this listserv if you want to talk about alpha fold really good things.

606

All right, thank you.

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Vocabulary and speaking notes for this lesson

This speaking lesson is built on the video “[TALK 13] Structure Prediction and Design using AlphaFold – Sami Chaaban”. The speaker keeps coming back to these words: protein, structure, alpha, fold, sequence. This video has 606 sentences and 7888 words to shadow. The speech runs for 41:41. The speaker talks fast, about 189 words per minute, so expect linked and reduced sounds. Only 81% of the words are among the 3,000 most common in English, so the vocabulary is demanding.

Key vocabulary in this video

15 words from the video worth learning, with pronunciation and meaning:

Word	Pronunciation	Meaning
alpha noun	/ˈæl.fə/	The name of the first letter of the Greek alphabet (Α, α), followed by beta. In the Latin alphabet it is the predecessor to A.
fold verb	/ˈfəʊld/	To bend (any thin material, such as paper) over so that it comes in contact with itself.
sequence noun	/ˈsiː.kwəns/	A set of things next to each other in a set order; a series.
prediction noun	/pɹɪˈdɪkʃən/	A statement of what will happen in the future.
predict verb	/pɹɪˈdɪkt/	To make a prediction: to forecast, foretell, or estimate a future event on the basis of knowledge and reasoning; to prophesy a future event on the basis of…
essentially adverb	/ɪˈsɛnʃəli/	In essence: by nature.
residue noun	/ˈɹɛzɪduː/	That which persists or remains following the removal or elimination of other elements.
interaction noun	/ˌɪn.təˈɹæk.ʃən/	The situation or occurrence in which two or more things act upon one another to produce an effect; the effect resulting from such a situation or occurrence.
alignment noun	/əˈlaɪn.mənt/	An arrangement of items in a line.
domain noun	/ˌdəʊˈmeɪ̯n/	A geographic area owned or controlled by a single person or organization.
bind verb	/ˈbaɪ̯nd/	To tie; to confine by any ligature.
coil noun	/kɔɪl/	Something wound in the form of a helix or spiral.
rank noun	/ˈɹæŋk/	A row of people or things organized in a grid pattern, often soldiers.
database noun	/ˈdeɪtəˌbeɪs/	A collection of (usually) organized information in a regular structure, usually but not necessarily in a machine-readable format accessible by a computer.
generate verb	/ˈd͡ʒɛn.ə.ɹeɪt/	To bring into being; give rise to.

Phrasal verbs you will hear

Word	Pronunciation	Meaning
come out verb	/ˌkʌm ˈaʊt/	To be discovered; to be revealed.
check out verb		To record the departure or withdrawal of someone or something (such as guests, employees, books, etc.).
get out verb		To leave or escape.
go through verb		To travel from one end of something to the other.
turn out verb		To end up; to result.
come out with verb		To say (something) unexpectedly.
figure out verb		To come to understand; to discover or find a solution; to deduce.
go ahead verb	/ˌɡoʊ əˈhɛd/	To proceed with; to begin.

Sentences worth repeating

Short, complete lines from the video that you can reuse in everyday conversation:

How do I cut my proteins so that I can purify better?
I'm not going to get too deep into it.
You'll get your multiple sequence alignment.
I'll talk about it in a second.
I mean, they're very far apart.

Grammar in this video

The structures the speaker uses most, with the exact words from the video:

Structure	In the video
Present perfect continuous have/has been + -ing — an action that started earlier and is still going on	we've been talking
Present perfect have/has + past participle — a past action that still matters now	have been · has been monitored · we've now come
Passive voice be + past participle — the focus is on what happens, not who does it	can be tested · has been monitored · can be predicted
Relative clauses who / which + clause — extra information about a person or thing	Baker, which is · algorithm, which is · notebook, which is

Pronunciation to watch

The speaker uses 149 contractions and reduced forms, such as you're, you'll, we're. Say them the short way, as you hear them.

The “sh” and “zh” sounds: prediction /pɹɪˈdɪkʃən/, essentially /ɪˈsɛnʃəli/, interaction /ˌɪn.təˈɹæk.ʃən/, structural /ˈstɹʌk(t͡)ʃəɹəl/, confirmation /ˌkɑn.fəɹˈmeɪ.ʃən/
Long words — get the stress right: essentially /ɪˈsɛnʃəli/, interaction /ˌɪn.təˈɹæk.ʃən/, necessarily /ˌnɛs.əˈsɛɹ.ə.li/, confirmation /ˌkɑn.fəɹˈmeɪ.ʃən/, speculation /ˌspɛk.jəˈleɪ.ʃən/

How to practise with this video

Listen to the whole video once without speaking and note the words you do not know.
Start at 0.75× speed, shadow it sentence by sentence, then go back to normal speed once it feels easy.
Record yourself and compare with the original, paying attention to words like alpha, fold, sequence.

What is the Shadowing Technique?

Shadowing is a science-backed language learning technique originally developed for professional interpreter training and popularized by polyglot Dr. Alexander Arguelles. The method is simple but powerful: you listen to native English audio and immediately repeat it out loud — like a shadow following the speaker with just a 1–2 second delay. Unlike passive listening or grammar drills, shadowing forces your brain and mouth muscles to simultaneously process and reproduce real speech patterns. Research shows it significantly improves pronunciation accuracy, intonation, rhythm, connected speech, listening comprehension, and speaking fluency — making it one of the most effective methods for IELTS Speaking preparation and real-world English communication.

Shadowing technique: read the full step-by-step guide →

Shadowing Practice: [TALK 13] Structure Prediction and Design using AlphaFold – Sami Chaaban - Learn English Speaking with Video

Choose a practice mode

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Vocabulary and speaking notes for this lesson

Key vocabulary in this video

Phrasal verbs you will hear

Sentences worth repeating

Grammar in this video

Pronunciation to watch

How to practise with this video

What is the Shadowing Technique?