00:02
Hi everyone, Susume here.  I'm talking about how to find shapes in the Foldit electron density cloud.
00:09
And we are looking at puzzle number 1152.
00:13
Just a quick review of the electron density menu settings:  I've got my threshold set right above the "M" in the word "mouse,"
00:21
and I've got my alpha, which is transparency, set a little bit to the right of center.
00:27
And I was most recently looking at backbone shapes, so I was using the solid cloud,
00:32
But I'm going to look for sidechains now, and I'm going to wireframe, 
00:36
I just find they show up better in the wireframe view.
00:40
Now we've got way too much cloud on the screen, it just looks like  a big blob, so I want to zoom in to the middle of the cloud.  
00:47
I have put my protein right through the middle of the cloud, and that's what's going to give us visibility in the middle.
00:52
I'm going to put my mouse on the backbone and hit shift-Q.
1:00
That's going to zoom us in.
1:05
And I have found some sheets in this cloud, and I marked the endpoints of the sheets with red.
1:13
There's a sheet right here going up and down, and that's the fist place that I'm going to look for sidechains.
1:19
I've got the protein near the sheet, I'm zoomed in, and I'm going to cut away.
1:25
Actually, I'm looking at the front of the sheet right now, and the sidechains are facing towards me and away from me,
1:31
which makes them hard to see, so I'm going to turn this 90 degrees so that I'm looking at the sheet in silhouette.
1:40
OK, I'm going to cut away some of the foreground and background, there's still too much cloud on the screen for me to see things clearly.
1:47
To cut away the foreground I'm going to use ctrl-alt-drag with the mouse. 
1:54
And to cut away the background I'm going to use ctrl-shift-drag.
2:05
And I can see this sheet clearly now, running from the red dot to the other red dot, going up and down.  It's kind of zigzag-y shaped.
2:13
And the sidechains are going off to the left and the right.  When I'm looking for sidechains to use as landmarks,
2:20
I always try to find the large ones with the rings on them.  The large orange sidechains with rings, they're called aromatics.
2:28
And those make the best landmarks.  Not every protein has those; this protein happens to have a lot of them.
2:35
And I can see the sidechain going off to the left here has a large head on it; it's kind of a wedge-shaped head.
2:41
It's got a skinny neck where it attaches to the backbone.  
2:46
This wedge-shaped head is going to be the kind of sidechain that has two rings together, a little ring here and a big ring there.
2:53
And I'm just going to mark that with a purple dot, so that I can find it later.
2:58
Moving down the sheet, we've got another sidechain on the right here that has a big round head, a narrow neck, and it attaches to the backbone.
3:07
I'm going to mark that with a purple dot.  And I notice that this round head does not have a tip on it.
3:14
That's important for figuring out what kind of sidechain it is.
3:18
Moving a little further down the sheet, I've got a Y-shaped sidechain.  It's a short one.  And I'm going to mark that with a green dot.
3:28
First I put a purple dot by hitting Tab.  Now the purple dot's hard to see, because it's in the foreground, and I cut away so much foreground.
3:36
So I'm just going to roll that piece of the protein back a little bit, towards the back of the screen, and then my dot becomes visible.
3:43
Put my mouse on the dot, right-click, and I can choose a new color for the dot.  OK.
3:52
And then moving up the sheet, the next sidechain is just a peg.  It doesn't have a head at all.
3:59
OK, I'm going to mark that one with a green dot as well.  
4:06
And I accidentally marked the wrong spot.  OK, I'm going to hit Tab again on that dot.  
4:12
There's a little green recycle bin here on the Point Information menu that'll get rid of the dot.
4:19
And let me see if I can put my dot on that peg.  There we go, and right click to choose the color.
4:27
OK, so I've got two purple dots and two green dots.  Those are marking sidechains on this one sheet.
4:35
And I'm going to flip now over to a text document that I use when I'm working on an electron density puzzle.
4:42
I went to the puzzle page on the website, and I got the sequence.  That's the list of letters that stand for the amino acids in the protein.
4:54
And since this is a long protein, I broke that sequence up into two lines, and I put it in my text document.
5:00
Then above the sequence, I typed in the segment numbers going from 1 all the way to 199.
5:09
And those are going to help me find things in the protein.
5:12
Then below the sequence, I typed in the secondary structure that was predicted on the protein.  
5:19
When we were given the protein, it had helixes and sheets already predicted on it.
5:24
These dashes are loops, the H's are helix segments, and the E's are sheets.  And again, I got those off the protein in the puzzle.
5:39
Then I made some notes down at the bottom about the kind of sidechains that I'm looking for as landmarks.
5:45
The double ring sidechain is abbreviated with a W.  And I got these letters off of the wiki.  
5:52
The big, single ring, without a tip on it, is abbreviated with an F.  
5:58
So I'm going to look for a spot where there's a W and an F next to each other in the sequence.  
6:03
They might go W, F; they might go F, w, because we're not sure which direction that sheet was facing.
6:10
OK, and it turns out in this protein there's only one place where a W and an F occur next to each other, and that's right here.
6:17
And that happens to be in the middle of a sheet, which is good because that's what we were looking at.
6:22
And the W is segment number 151.  So we're going to go find segment number 151 and move it into the cloud,
6:32
in that exact spot, and see if we can get the W and F sidechains to match up with their spots in the cloud.
6:40
OK I'm going to turn this back 90 degrees.  I happen to have this lined up, 
6:47
so that segment 151 is right here.  I'm going to hit Tab on it.  
6:52
It's a tryptophan, which is what's abbreviated with a W.  You can see it's a double ring.
6:57
And let's see if we can rotate.  Its neighbor, right here, is the large single ring.  
7:03
OK, I'm going to move those into the cloud.
7:06
If you're ever trying to use the move tool in an electron density puzzle, and you can't see the move tool, 
7:12
it might be because the move tool lives in the foreground.  And if you have cut away much of the foreground, your move tool might not be visible.
7:21
If that's the case, you can increase the foreground visibility until that move tool reappears.  
7:28
OK, we're going to slide that into the cloud.  
7:35
And put the focus on it and hit shift-Q.
7:42
Going to turn it so we can see the sidechains again, and we're goign to cut away some foreground, using ctrl-alt-drag.
7:52
Cut away some background using ctrl-shift-drag.  
7:58
OK, I've actually got another sheet here in front of the sheet I want to look at.
8:04
So I'm going to try changing the focus of the camera.  Put my cursor right on the part that's in the middle of the sheet, and hit shift-Q again.
8:13
Changes the zoom.  And let's cut away some foreground, ctrl-alt-drag.  
8:22
Cut away some background, ctrl-shift-drag.
8:27
There we go, now I'm looking at the sheet I want to see, the one with the dots on it.
8:32
OK, I want the double ring to be on the left, single ring on the right, and I want the double ring to be above the single ring.
8:40
Let's see if we can move the protein that way.  OK, I lost my move tool; I'm going to increase foreground visibility with ctrl-alt-drag,
8:50
until I can see the move tool.  
8:57
See if we got this the right way. Lost my move tool again, that's frustrating.
9:04
And I've got the double ring on the bottom and the single ring on the top, that's the opposite of what I want, so I'm going to flip this around.
9:17
There we go.  OK, and I'm going to check it from 90 degrees. 
9:25
Get it lined up a little better with the backbone.  Flip back 90 degrees.
9:35
See if I can get those sidechains right in their pockets.  OK, I've got the big ring in its pocket.
9:41
The little Y, down here, is almost in its pocket; I need to move down a little bit.  OK, that's lined up pretty well.
9:48
Now, this double ring up here is flipped the wrong way.  I'm just going to give that a tug, and it will flip down into the right position.
9:57
And then the fourth one we found is this little peg up here, and it's in its pocket.  So I've got those lined up pretty well.
10:05
I am going to put bands on those.  I like to put a band at the first corner in the sidechain.
10:13
When I place a band, I like to use the Alt key, in addition to shift-mouse.  The alt key keeps the band from sticking to any other part of the protein.
10:24
Woops.  OK, I'm banding all four of the ones that I placed in the cloud.
10:33
The bands, at this point, are just acting as markers for me, so I know that I've placed those sidechains where I want them.
10:40
Once I've placed as much of the protein in the cloud as I can, before I start my first wiggle, I'm going to increase the strength on those bands to 10.
10:51
And that's going to help hold the protein in the cloud during those first wiggles and shakes when it's getting settled.
10:58
OK, I've got my bands marking my sidechains.  I'm going to get rid of these dots now, 
11:04
so that when I make new purple and green dots elsewhere, I don't get mixed up about which ones I'm currently working with.
11:16
Now I notice, as I move down the sheet, that there is another large ring sidechain down here at the bottom.  And this one has a tip on it.
11:26
So I definitely want to find the place in the cloud where that large sidechain is going to fit.
11:31
I'm just going to increase the foreground visibility a bit, so I can see more cloud.
11:38
And I can see that right now the sidechain is pointing off into an empty space.  There's no cloud there.  Let me spin this around.
11:46
And there it is.  I see the pocket.  Here's the wide part; here's the neck.  You can see the neck connecting to the backbone.
11:56
And this one has a really big tip on it.  In fact, the tip on this is so long that it seems to be intersecting with another part of the protein.
12:04
I'm just going to bump the threshold up a little bit, and make sure that that detaches when there's a higher threshold.
12:13
There we go.  Now the tip has detached, and we have a nice view of the ring and the tip on that sidechain.
12:20
So I definitely want to fit that sidechain in there.  
12:23
And I notice that the backbone coming off of that sidechain is curled.  It looks like a little bit of helix there.
12:31
So I'm going to make a helix to fit in there.  I'm going to cut the backbone, give myself, I don't know, four segments.
12:41
And we'll change that to helix.  
12:49
And curl it up.
12:55
OK, and I want to fit that into this piece of cloud.
13:00
Now I could have banded the sidechain to there and used rebuild, but in this case, since it looked like helix,
13:11
I decided to go ahead and make a helix to fit in there.
13:25
OK, I've got that sidechain fit in pretty well.  I've got the backbone fitting into the curl.
13:36
It's important to fit your sidechains into the cloud as well as you can.  
13:40
If you just get the backbone in the cloud, and you don't get the sidechains onto the right side, 
13:46
you're going to suffer in your density scores.  If you want to get the really high density scores on the segments, 
13:52
you need to get the sidechains into their pockets in the cloud.  OK, I'm going to band that.  
13:59
And then, in order to see more backbone, I would have to lower the threshold again.
14:05
But that's a look at how we do the sidechains.  Use them as landmarks to tell you where the backbone needs to go.
14:12
And in the next video we will look at how to find helixes.  And thanks everyone for watching!