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EDWARD BRIGNOLE: My name's Ed.

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I'm a postdoc in
Cathy Drennan's lab,

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and previously, I had worked at
the Scripps Research Institute

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with Francisco Asturias.

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So some of the work
that I did there

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is what Liz and Joanne like,
and we'll talk about that.

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So I thought I'd start with
just finding out, has anybody

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00:00:47,830 --> 00:00:50,490
here done electron microscopy.

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You've done some EM.

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00:00:51,570 --> 00:00:54,343
OK, on--

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AUDIENCE: Gold nanoparticles
with a [INAUDIBLE]

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spirit thing.

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EDWARD BRIGNOLE: OK.

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Over here?

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AUDIENCE: No.

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00:01:01,664 --> 00:01:02,514
In St. Louis.

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EDWARD BRIGNOLE:
In St. Louis, OK.

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00:01:03,930 --> 00:01:06,840
So you sat at the microscope
and worked on obs and--

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AUDIENCE: That was my
favorite [INAUDIBLE]

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EDWARD BRIGNOLE: Yeah.

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Anybody else?

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00:01:13,230 --> 00:01:16,440
So how about a light microscope?

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You guys used?

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00:01:17,670 --> 00:01:19,560
High school biology, maybe?

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OK.

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00:01:20,060 --> 00:01:21,690
Everybody's used a
light microscope.

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All right.

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00:01:22,189 --> 00:01:24,210
So that's good.

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00:01:24,210 --> 00:01:25,950
And then I guess at
this point, you guys

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00:01:25,950 --> 00:01:29,445
have had two lectures
on fatty acid synthesis,

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00:01:29,445 --> 00:01:31,980
so you sort of have some
feel for the enzymes

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00:01:31,980 --> 00:01:33,900
and who's involved
and what they do.

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00:01:33,900 --> 00:01:36,000
All right, so I thought
we'd spend the first 20

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minutes talking about electron
microscopy and what it can do

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and how it could be used.

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And there is
actually quite a bit

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that's changed since
this paper in 2009.

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00:01:45,780 --> 00:01:49,300
There's a lot that's happened
in the last few years.

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00:01:49,300 --> 00:01:51,240
And we can talk
briefly about that.

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00:01:51,240 --> 00:01:53,850
And then we can move into
fatty acid biosynthesis

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00:01:53,850 --> 00:01:56,730
and tie that into what you
guys have learned already.

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00:01:56,730 --> 00:01:59,940
And then there was a
bonus paper at the end.

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00:01:59,940 --> 00:02:01,860
If you are really
excited about this,

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00:02:01,860 --> 00:02:05,400
there's some polyketide
synthase structures

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that have come out in
the last year or two.

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And those are
pretty interesting.

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So if you've got the handout
handy, there's some questions.

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These are what I thought we'd
focus the conversation around.

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The first part's about
fatty acid synthase in EM,

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and then there's these
bonus ones at the end.

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00:02:29,040 --> 00:02:32,700
So when you guys were looking
in the light microscope,

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you're probably looking
at biological samples,

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I would guess.

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So you were probably
looking at, say, a cell.

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But all the bits and
pieces of the cell

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that perform all these
interesting functions,

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we want to understand these.

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And so being able to actually
see them and see them in action

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allows us to understand
how they work.

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So for instance, if you pick out
this piece of machinery here--

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and you can see that
it's got an active site

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where it binds substrate
and maybe moves it around

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or acts on it in some way.

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And you might have
allosteric subunits,

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and you can find that it's
got four subunits that

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are round like wheels.

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And it can move from
one place to the other.

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This is just an
analogy, but same thing

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would go for, say, motor
proteins transporting cargo,

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or in this case,
fatty acid synthase.

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So if you've used
a light microscope,

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the electron microscope is
conceptually very similar.

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You've got the light source
at the top versus an electron

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source.

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Condenser lens will focus
that on the specimen.

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The objective lens
forms the image.

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It's magnified by the projector
lens, and what you get out

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is this enlarged image.

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So you can see things that
you couldn't see by eye.

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Light microscope, you can
get up to about 1,000x.

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And in an electron microscope,
you can go up to 500,000x

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or even beyond that.

90
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So maybe an interesting
place to start here

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is why can electron
microscopes do this

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but light microscopes that.

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Why can you get only
this magnification

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00:04:04,950 --> 00:04:06,288
with a light microscope?

95
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Guesses?

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00:04:10,442 --> 00:04:13,480
If you look back up at the
top, what are the sources?

97
00:04:13,480 --> 00:04:16,756
You're using light
versus electrons.

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Why would you be able to get
a higher magnification image

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00:04:20,589 --> 00:04:24,135
using electrons than light?

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AUDIENCE: Diffraction limit.

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00:04:25,940 --> 00:04:26,856
EDWARD BRIGNOLE: Yeah.

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Do you know why?

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Why would light have
a diffraction limit

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00:04:35,680 --> 00:04:38,220
that's in the micron
range or nanometer

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00:04:38,220 --> 00:04:43,850
range versus electrons in the
actually like picometer range?

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AUDIENCE: Since your
wavelength could be--

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EDWARD BRIGNOLE: Exactly.

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That's what I was looking for.

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00:04:47,520 --> 00:04:49,630
Yeah, wavelength is
what I was looking for.

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00:04:49,630 --> 00:04:55,470
So either visible or even if
you go to a UV light source,

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00:04:55,470 --> 00:04:59,770
you're talking about
nanometer-sized waves

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00:04:59,770 --> 00:05:03,000
versus electrons, at the
typical electron acceleration

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00:05:03,000 --> 00:05:04,890
voltages that are
used, are in the tens

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00:05:04,890 --> 00:05:07,600
of picometer wavelength.

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00:05:07,600 --> 00:05:09,520
And so that's the main reason.

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00:05:09,520 --> 00:05:11,160
You can magnify
these images further.

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00:05:11,160 --> 00:05:14,430
But you're not going to
get any higher resolution

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versus an electron microscope.

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00:05:18,900 --> 00:05:20,640
But you don't often
hear about tens

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of picometer resolution
images by EM.

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00:05:22,800 --> 00:05:27,060
So I guess maybe I'll
flip back to this slide.

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00:05:27,060 --> 00:05:30,530
So this is differences
in the source.

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00:05:30,530 --> 00:05:32,250
But you could
actually theoretically

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00:05:32,250 --> 00:05:37,020
go 100 times or more beyond
these magnifications by EM.

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00:05:37,020 --> 00:05:39,510
Why do you not typically
hear about that?

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00:05:39,510 --> 00:05:42,420
What else could be
limiting resolution

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00:05:42,420 --> 00:05:44,250
as you go down through
this path here?

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00:05:51,362 --> 00:05:52,570
A number of you wear glasses.

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00:05:52,570 --> 00:05:54,620
Do they perfectly
correct your vision?

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00:05:54,620 --> 00:05:55,787
They don't for me.

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00:05:55,787 --> 00:05:57,370
Yeah, so same thing
with these lenses.

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No lens is perfect, and you've
got different aberrations.

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So the way light or
electrons that are coming in

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are bent is never perfect.

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They're not going to achieve--

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00:06:10,800 --> 00:06:15,700
and there's correctors that you
can use to compensate for this.

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00:06:15,700 --> 00:06:17,530
Also the wavelength
of the light,

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having it perfectly tuned
to a particular energy

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00:06:21,130 --> 00:06:22,910
of either photons
or electrons, there

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00:06:22,910 --> 00:06:25,079
is going to be
some distribution.

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00:06:25,079 --> 00:06:26,620
And so you're going
to have some that

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are a little more
redshifted or blueshifted,

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00:06:29,000 --> 00:06:30,250
higher energy or lower energy.

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And those are going to also not
come to complete focus there.

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00:06:34,100 --> 00:06:38,320
And so this is for the
lenses in the source, why

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00:06:38,320 --> 00:06:41,440
you would typically be limited
to about an angstrom resolution

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00:06:41,440 --> 00:06:45,190
unless you buy some fancy
correctors for your microscope

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00:06:45,190 --> 00:06:48,040
to correct for spherical
aberration or energy filters

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00:06:48,040 --> 00:06:50,252
to correct for
chromatic aberration.

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00:06:55,080 --> 00:06:56,740
So what kinds of
things in the cell

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00:06:56,740 --> 00:06:58,840
could we look at by
electron microscopy?

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Maybe you guys have
seen images in papers,

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00:07:05,790 --> 00:07:10,570
probably in your
textbooks, EM images of--

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what?

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00:07:15,370 --> 00:07:15,970
Help me out.

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You see in sections about,
say, muscle where there's

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a section of some muscle
fiber where you can actually

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see some of the proteins that
are involved, the filaments.

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So you've seen things like that.

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Tissue sections.

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00:07:34,330 --> 00:07:36,020
You could look at
tissue sections by EM.

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00:07:36,020 --> 00:07:39,220
You could look at
individual cells.

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Could you look at an elephant
by electron microscopy?

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00:07:42,580 --> 00:07:43,920
Have you ever seen that?

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00:07:43,920 --> 00:07:44,770
No.

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00:07:44,770 --> 00:07:47,690
So why would you not
image an elephant

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in an electron microscope?

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00:07:49,798 --> 00:07:52,220
AUDIENCE: Simply because
they're too [INAUDIBLE]..

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00:07:52,220 --> 00:07:53,931
EDWARD BRIGNOLE: OK, yeah.

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Exactly.

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00:07:54,430 --> 00:07:56,590
You'd have a really hard
time preparing that elephant

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even if you had a microscope
that was big enough.

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00:08:01,119 --> 00:08:03,160
But you could, say, take
an x-ray of an elephant.

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00:08:03,160 --> 00:08:03,910
Right?

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00:08:03,910 --> 00:08:06,190
But what is it about
electrons, maybe,

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00:08:06,190 --> 00:08:10,520
that you wouldn't have
to deal with with x-rays?

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00:08:10,520 --> 00:08:12,437
AUDIENCE: Killing the elephant?

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00:08:12,437 --> 00:08:13,478
EDWARD BRIGNOLE: I mean--

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00:08:13,478 --> 00:08:17,950
[LAUGHTER] Right.

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00:08:17,950 --> 00:08:21,370
So why would the elephant
have to be dead to image it

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00:08:21,370 --> 00:08:23,968
in an electron microscope?

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00:08:23,968 --> 00:08:25,952
AUDIENCE: When
you're shooting it

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00:08:25,952 --> 00:08:30,482
with electrons, even just for
the cell, it'll kill the cell.

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00:08:30,482 --> 00:08:32,177
[INAUDIBLE] you do it to a--

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00:08:32,177 --> 00:08:32,676
Oh, wait.

186
00:08:32,676 --> 00:08:35,285
Don't you have to [INAUDIBLE]?

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00:08:35,285 --> 00:08:36,201
EDWARD BRIGNOLE: Yeah.

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00:08:36,201 --> 00:08:39,490
So in some cases, you
might negatively stain.

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00:08:39,490 --> 00:08:43,084
Typically, you would stain a
sample in some way or another.

190
00:08:43,084 --> 00:08:44,750
But what I was getting
at is the vacuum.

191
00:08:44,750 --> 00:08:47,350
So the microscope is
under high vacuum.

192
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So electrons have
mass, and they're

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going to interact
strongly with the matter

194
00:08:50,560 --> 00:08:51,684
that they're going through.

195
00:08:51,684 --> 00:08:54,710
You actually couldn't get an
electron through an elephant.

196
00:08:54,710 --> 00:08:59,015
You could get x-rays
through an elephant though.

197
00:08:59,015 --> 00:09:00,490
So thickness is one issue.

198
00:09:00,490 --> 00:09:03,660
You'd have to cut really thin
sections of your elephant,

199
00:09:03,660 --> 00:09:05,470
so about 200 nanometers thick.

200
00:09:05,470 --> 00:09:08,380
You can go thicker than
that, but then there

201
00:09:08,380 --> 00:09:10,870
are some other issues with
resolution that occur.

202
00:09:10,870 --> 00:09:14,740
So this is about the high
end of what you would want

203
00:09:14,740 --> 00:09:17,890
to be for a good EM specimen.

204
00:09:17,890 --> 00:09:24,850
And then if we're looking at,
say, individual macromolecules,

205
00:09:24,850 --> 00:09:26,470
the size of those
macromolecules,

206
00:09:26,470 --> 00:09:29,106
to be able to look at the
image and pick them out,

207
00:09:29,106 --> 00:09:31,480
would have to be-- if there
are single particles floating

208
00:09:31,480 --> 00:09:33,340
around like a virus
particle or something,

209
00:09:33,340 --> 00:09:34,340
you can usually do that.

210
00:09:34,340 --> 00:09:36,460
Because they're much bigger
than 100 kilodaltons.

211
00:09:36,460 --> 00:09:38,630
Probably in your textbook,
you've seen EM images.

212
00:09:38,630 --> 00:09:40,887
Or even in newspapers, you
pick up the New York Times,

213
00:09:40,887 --> 00:09:42,970
and there's an article on
Zika virus or something,

214
00:09:42,970 --> 00:09:45,890
and there's an EM image of it.

215
00:09:45,890 --> 00:09:49,350
So those are much bigger
than 100 kilodaltons.

216
00:09:49,350 --> 00:09:53,470
But this is about the lower
end for individual particles.

217
00:09:53,470 --> 00:09:55,720
So I thought I'd
just throw this up

218
00:09:55,720 --> 00:09:58,474
so you could look at
the different bits

219
00:09:58,474 --> 00:10:00,265
and pieces of an ant
in a light microscope.

220
00:10:00,265 --> 00:10:03,160
An electron microscope
largely overlaps

221
00:10:03,160 --> 00:10:05,500
with the high end of the
light microscope, where

222
00:10:05,500 --> 00:10:08,230
you could look at cells
or sections of cells

223
00:10:08,230 --> 00:10:11,350
if the cell is a
micron or more thick.

224
00:10:11,350 --> 00:10:16,710
Bacteria, bacteria-like viruses,
bacteriophage in this case.

225
00:10:16,710 --> 00:10:19,429
Electron microscopy has
resolution down to this range.

226
00:10:19,429 --> 00:10:21,220
But in order to visualize
things like this,

227
00:10:21,220 --> 00:10:23,620
you'd have to be able to
pick them out of your image.

228
00:10:23,620 --> 00:10:27,280
So you could assemble 10
kilodalton particles into,

229
00:10:27,280 --> 00:10:30,310
say if it's actin or something,
into a large polymer.

230
00:10:30,310 --> 00:10:31,990
Then you can pick
out the polymer,

231
00:10:31,990 --> 00:10:34,180
and in the process
of reconstructing it,

232
00:10:34,180 --> 00:10:36,674
identify, say, 10
kilodalton-sized subunits.

233
00:10:36,674 --> 00:10:39,340
But to look through an image and
pick out a 10 kilodalton piece,

234
00:10:39,340 --> 00:10:41,410
that would be impossible.

235
00:10:41,410 --> 00:10:45,730
And then x-ray
crystallography and NMR

236
00:10:45,730 --> 00:10:49,870
are typically imaging
structures of about this size

237
00:10:49,870 --> 00:10:53,301
down to resolutions
in the angstrom range.

238
00:10:53,301 --> 00:10:53,800
All right.

239
00:10:53,800 --> 00:10:56,380
So we've sort of gone
through the different kinds

240
00:10:56,380 --> 00:10:58,440
of things you can see.

241
00:10:58,440 --> 00:11:00,430
And then the one last
thing I wanted to say

242
00:11:00,430 --> 00:11:03,430
is, there's lots of different
kinds of cellular structures

243
00:11:03,430 --> 00:11:06,020
you can look at in an
electron microscope.

244
00:11:06,020 --> 00:11:09,040
And I hinted at, say,
if you can assemble

245
00:11:09,040 --> 00:11:11,400
smaller pieces into
larger structures,

246
00:11:11,400 --> 00:11:12,400
then you can image them.

247
00:11:12,400 --> 00:11:16,360
So if you can coax, say, a
G protein-coupled receptor

248
00:11:16,360 --> 00:11:20,590
that you're interested in
into a two-dimensional array,

249
00:11:20,590 --> 00:11:24,940
then you could visualize these
small very interesting proteins

250
00:11:24,940 --> 00:11:27,640
as part of this 2D
crystalline array.

251
00:11:27,640 --> 00:11:30,640
Or in the case of actin,
polymerized into a filament.

252
00:11:30,640 --> 00:11:33,680
And so there are different
ways to reconstruct molecules

253
00:11:33,680 --> 00:11:36,430
that arrange themselves into
arrays by, say, electron

254
00:11:36,430 --> 00:11:41,620
diffraction or in filaments
because each unit is related

255
00:11:41,620 --> 00:11:45,350
to the unit that's before
and after it in the filament.

256
00:11:45,350 --> 00:11:46,879
But the brand of
electron microscopy

257
00:11:46,879 --> 00:11:48,670
I'm largely going to
be talking about today

258
00:11:48,670 --> 00:11:50,170
is what we call
single particle EM,

259
00:11:50,170 --> 00:11:53,320
where you've got these
freestanding proteins or virus

260
00:11:53,320 --> 00:11:54,967
particles in solution.

261
00:11:54,967 --> 00:11:57,550
And you're going to try to pick
individual ones out and figure

262
00:11:57,550 --> 00:11:59,860
out what their 3D structure is.

263
00:11:59,860 --> 00:12:03,400
And each molecule is independent
and not necessarily related

264
00:12:03,400 --> 00:12:05,720
to the other ones
that are around it.

265
00:12:09,260 --> 00:12:09,920
All right.

266
00:12:09,920 --> 00:12:13,910
So we talked a little bit
about why an elephant wouldn't

267
00:12:13,910 --> 00:12:15,570
survive in the microscope.

268
00:12:15,570 --> 00:12:18,110
And that had to do with
specimen preparation.

269
00:12:18,110 --> 00:12:21,260
So electrons, because
they scatter strongly off

270
00:12:21,260 --> 00:12:23,960
of the matter that
it's traveling through,

271
00:12:23,960 --> 00:12:26,675
if you have gas in your column.

272
00:12:26,675 --> 00:12:29,300
Then the electrons are going to
scatter off of that before they

273
00:12:29,300 --> 00:12:30,133
get to your protein.

274
00:12:30,133 --> 00:12:33,410
So the really good microscopes
have really high vacuums,

275
00:12:33,410 --> 00:12:35,660
and the specimen has
to be preserved somehow

276
00:12:35,660 --> 00:12:36,710
to survive that.

277
00:12:36,710 --> 00:12:41,030
So you probably wouldn't want to
just put your protein in buffer

278
00:12:41,030 --> 00:12:43,434
and stick it into the
microscope because basically,

279
00:12:43,434 --> 00:12:45,350
all the buffer would
evaporate, and you'd just

280
00:12:45,350 --> 00:12:46,810
have a dried out protein.

281
00:12:46,810 --> 00:12:48,920
So you need some way
to either, if you're

282
00:12:48,920 --> 00:12:51,080
going to dehydrate
it, to stain it,

283
00:12:51,080 --> 00:12:53,960
which is what we're going
to talk about in this paper.

284
00:12:53,960 --> 00:12:57,590
Or you can cryogenically
preserve it and then

285
00:12:57,590 --> 00:12:59,480
keep it at liquid
nitrogen temperatures

286
00:12:59,480 --> 00:13:02,020
while you're imaging it.

287
00:13:02,020 --> 00:13:04,870
And then I think
one of you guys also

288
00:13:04,870 --> 00:13:08,140
mentioned radiation damage, that
the elephant wouldn't survive

289
00:13:08,140 --> 00:13:09,880
being bombarded by radiation.

290
00:13:09,880 --> 00:13:14,410
And so at the specimen level,
these radiation damage doses

291
00:13:14,410 --> 00:13:18,550
are equivalent to an atomic bomb
going off if you scale it up.

292
00:13:22,760 --> 00:13:25,261
And so basically, this is what
you're doing to your specimen

293
00:13:25,261 --> 00:13:26,260
while you're imaging it.

294
00:13:26,260 --> 00:13:28,780
And so in your case, you're
looking at gold nanoparticles,

295
00:13:28,780 --> 00:13:29,321
you had said.

296
00:13:29,321 --> 00:13:32,650
And so you can hit a hefty
dose on a gold nanoparticle.

297
00:13:32,650 --> 00:13:34,630
But on biological
specimen, you'd

298
00:13:34,630 --> 00:13:39,490
be breaking carbon-carbon
bonds, and your protein

299
00:13:39,490 --> 00:13:42,940
is rupturing as
you're imaging it.

300
00:13:42,940 --> 00:13:45,070
So typically in
electron microscopy,

301
00:13:45,070 --> 00:13:47,620
we'll only expose
the area that we're

302
00:13:47,620 --> 00:13:49,810
going to-- and for
biological specimens,

303
00:13:49,810 --> 00:13:52,510
just as we'll focus adjacent
where we're actually

304
00:13:52,510 --> 00:13:54,440
going to expose and
then expose the area.

305
00:13:54,440 --> 00:13:57,100
So the first time that area
sees a decent dose of electrons

306
00:13:57,100 --> 00:14:00,577
is when you're actually
acquiring an image of it.

307
00:14:00,577 --> 00:14:02,410
And I guess one last
thing I could point out

308
00:14:02,410 --> 00:14:04,450
about this 30 electrons
per angstrom squared

309
00:14:04,450 --> 00:14:07,360
is, even by 30 electrons
per angstrom squared dose

310
00:14:07,360 --> 00:14:11,080
on your specimen, a large amount
of the high resolution signal

311
00:14:11,080 --> 00:14:12,220
is already lost.

312
00:14:12,220 --> 00:14:15,160
So the first five electrons
per angstrom squared

313
00:14:15,160 --> 00:14:17,047
has most of the high
resolution information.

314
00:14:17,047 --> 00:14:18,880
But it doesn't have
enough information in it

315
00:14:18,880 --> 00:14:21,280
to actually visualize
your whole structure.

316
00:14:21,280 --> 00:14:22,840
So you want to
give it enough dose

317
00:14:22,840 --> 00:14:25,307
that you can see the whole
thing but not so much dose

318
00:14:25,307 --> 00:14:26,890
that you've destroyed
the whole thing.

319
00:14:26,890 --> 00:14:31,640
And I guess one other
thing that limits

320
00:14:31,640 --> 00:14:33,230
what we can see
in the microscope

321
00:14:33,230 --> 00:14:36,740
is, if you want to image
something at atomic resolution

322
00:14:36,740 --> 00:14:38,990
and the stage that's
holding the specimen

323
00:14:38,990 --> 00:14:41,430
is moving by a few
angstroms at the same time,

324
00:14:41,430 --> 00:14:43,220
then it's going to be blurry.

325
00:14:43,220 --> 00:14:46,790
The features you're looking
for are blurred out.

326
00:14:46,790 --> 00:14:49,469
I mentioned at the beginning
that this paper was in 2009

327
00:14:49,469 --> 00:14:50,760
that we're going to talk about.

328
00:14:50,760 --> 00:14:53,060
So in the last two
to three years,

329
00:14:53,060 --> 00:14:56,190
there's some new detectors
that have come online.

330
00:14:56,190 --> 00:14:58,830
And these are
revolutionizing the field.

331
00:14:58,830 --> 00:15:02,570
So if you look at structures
by single particle EM that

332
00:15:02,570 --> 00:15:05,390
are at less than five
angstroms resolution,

333
00:15:05,390 --> 00:15:07,910
it went from, around
the time of this paper,

334
00:15:07,910 --> 00:15:14,150
there were one or two to now
there are tens to even 100

335
00:15:14,150 --> 00:15:18,170
a year in the
last-- like in 2015.

336
00:15:18,170 --> 00:15:20,270
So there's a whole
mess of developments

337
00:15:20,270 --> 00:15:22,130
that are responsible
for this, but the one

338
00:15:22,130 --> 00:15:24,980
that's the most
important of these

339
00:15:24,980 --> 00:15:27,350
is these direct
electron detectors.

340
00:15:27,350 --> 00:15:30,949
So did you guys have
a chance to look at,

341
00:15:30,949 --> 00:15:32,990
say, the figures of the
paper we're going to talk

342
00:15:32,990 --> 00:15:34,036
about today?

343
00:15:34,036 --> 00:15:36,410
Did you have a chance to look
through the methods at all?

344
00:15:39,400 --> 00:15:43,220
Did anybody notice how
the images were acquired?

345
00:15:43,220 --> 00:15:45,320
So this predates direct
electron detectors.

346
00:15:45,320 --> 00:15:47,030
So what sort of
detectors were used?

347
00:15:51,130 --> 00:15:52,782
Anybody notice?

348
00:15:52,782 --> 00:15:55,330
Going once.

349
00:15:55,330 --> 00:15:59,910
OK, so some of the images
were collected on CCD cameras.

350
00:15:59,910 --> 00:16:03,140
And some were collected on film.

351
00:16:03,140 --> 00:16:10,900
So can you think of an advantage
of one versus the other?

352
00:16:10,900 --> 00:16:13,390
Anybody here into photography?

353
00:16:13,390 --> 00:16:15,140
Friends who are
into photography?

354
00:16:15,140 --> 00:16:17,770
Does anybody still shoot
their images on film?

355
00:16:20,490 --> 00:16:23,950
Maybe some purists of
image quality, something?

356
00:16:23,950 --> 00:16:28,140
Anyway, but why do most people
use digital cameras these days?

357
00:16:30,234 --> 00:16:32,400
You don't have to go and
develop your film, for one.

358
00:16:32,400 --> 00:16:33,370
Right?

359
00:16:33,370 --> 00:16:35,918
You probably don't even know
about having to go and develop,

360
00:16:35,918 --> 00:16:36,417
though.

361
00:16:39,519 --> 00:16:40,935
So that's a distinct
disadvantage,

362
00:16:40,935 --> 00:16:44,220
is the throughput.

363
00:16:44,220 --> 00:16:46,170
You can snap 100
pictures on your camera.

364
00:16:46,170 --> 00:16:48,657
You don't have to wait a couple
of days to see the results.

365
00:16:48,657 --> 00:16:50,740
So the same thing would
be an electron microscope.

366
00:16:50,740 --> 00:16:52,740
So if you're imaging
your specimen on film

367
00:16:52,740 --> 00:16:54,990
and then you have to take
the film cassette out and go

368
00:16:54,990 --> 00:16:56,340
into the darkroom
and develop your film

369
00:16:56,340 --> 00:16:58,470
and then realize that
there was some parameter

370
00:16:58,470 --> 00:17:00,881
wrong or somebody didn't
change the developer recently,

371
00:17:00,881 --> 00:17:02,130
the whole batch would be gone.

372
00:17:02,130 --> 00:17:07,050
So throughput with film is low,
but the signal-to-noise ratio

373
00:17:07,050 --> 00:17:12,150
and the point spread function
of detecting the electrons where

374
00:17:12,150 --> 00:17:14,500
they strike the film is good.

375
00:17:14,500 --> 00:17:17,940
So the image quality
is better with film.

376
00:17:17,940 --> 00:17:21,131
And also, the area that you
would expose is bigger on film

377
00:17:21,131 --> 00:17:21,630
too.

378
00:17:21,630 --> 00:17:25,770
So typical CCD cameras
are, say, 4K by 4K pixels.

379
00:17:25,770 --> 00:17:27,296
Film would be like 10K by 6K.

380
00:17:27,296 --> 00:17:28,920
So you'd have a much
bigger area, which

381
00:17:28,920 --> 00:17:31,765
means more particles per image.

382
00:17:31,765 --> 00:17:33,390
So that would be the
advantage of film.

383
00:17:33,390 --> 00:17:37,230
CCD cameras, I mentioned, are
a little worse performing.

384
00:17:37,230 --> 00:17:39,570
So what they have is
a scintillator layer.

385
00:17:39,570 --> 00:17:41,950
So it's like a phosphor layer.

386
00:17:41,950 --> 00:17:45,570
So the electrons would
come down, and some of them

387
00:17:45,570 --> 00:17:47,280
hit the scintillator
and bounce off.

388
00:17:47,280 --> 00:17:49,140
Some of them will hit
the scintillator layer

389
00:17:49,140 --> 00:17:50,340
and go through.

390
00:17:50,340 --> 00:17:52,440
Some of them will hit
the scintillator layer

391
00:17:52,440 --> 00:17:55,064
and zig around for a little bit
and then give off some photons.

392
00:17:58,110 --> 00:18:01,112
So you can see what
the disadvantages are

393
00:18:01,112 --> 00:18:02,820
if you're limited to,
say, a 30 electrons

394
00:18:02,820 --> 00:18:04,030
per angstrom squared dose.

395
00:18:04,030 --> 00:18:06,990
If a decent number of your
electrons are being lost

396
00:18:06,990 --> 00:18:09,210
or not detected
accurately-- in this case,

397
00:18:09,210 --> 00:18:11,796
you have a point
spread about the area

398
00:18:11,796 --> 00:18:13,170
where that electron
struck, where

399
00:18:13,170 --> 00:18:15,120
you're actually picking it up.

400
00:18:15,120 --> 00:18:16,620
So this is going
to cause a blurring

401
00:18:16,620 --> 00:18:18,990
of your high resolution signal.

402
00:18:18,990 --> 00:18:21,535
And then this is just to convert
the electrons to photons.

403
00:18:21,535 --> 00:18:23,160
Then you would
typically have some sort

404
00:18:23,160 --> 00:18:26,190
of fiber optic coupling, where
you would also lose some signal

405
00:18:26,190 --> 00:18:27,810
and also has a point spread.

406
00:18:27,810 --> 00:18:30,990
And then this is connected
to the actual detector,

407
00:18:30,990 --> 00:18:33,030
like what's in your
phone, basically.

408
00:18:37,085 --> 00:18:38,460
So this is how
some of the images

409
00:18:38,460 --> 00:18:39,930
were collected in the paper.

410
00:18:39,930 --> 00:18:42,180
Basically, the nice thing
about the CCD camera

411
00:18:42,180 --> 00:18:43,140
is its high throughput.

412
00:18:43,140 --> 00:18:45,460
You can get lots of
images really fast.

413
00:18:45,460 --> 00:18:48,690
But for the data that was
used to generate the 3D

414
00:18:48,690 --> 00:18:52,890
reconstructions, that
was collected on film.

415
00:18:52,890 --> 00:18:54,780
And then I guess I'll
just say one more

416
00:18:54,780 --> 00:18:57,480
word about then, these
direct electron detectors.

417
00:18:57,480 --> 00:19:00,600
Basically, they cut out
all this extra business.

418
00:19:00,600 --> 00:19:03,090
You basically detect
the electrons directly.

419
00:19:03,090 --> 00:19:07,310
So they come in, and actually,
each pixel has the ability--

420
00:19:07,310 --> 00:19:10,080
on some of these, the newest
top-of-the-line detectors,

421
00:19:10,080 --> 00:19:13,770
can actually figure out
which quadrant in the pixel

422
00:19:13,770 --> 00:19:18,390
the electron struck and can
actually count each electron

423
00:19:18,390 --> 00:19:21,330
event on each pixel
as it's happening.

424
00:19:21,330 --> 00:19:23,970
So you have some
electronic noise in here,

425
00:19:23,970 --> 00:19:25,380
so there's little bits of noise.

426
00:19:25,380 --> 00:19:27,480
And on a typical CCD
camera, you would

427
00:19:27,480 --> 00:19:31,730
integrate this whole signal
over time to come up with--

428
00:19:31,730 --> 00:19:34,120
this is down here.

429
00:19:34,120 --> 00:19:38,550
So you'd integrate the charge
that's accumulated over time.

430
00:19:38,550 --> 00:19:40,050
But in these counting
detectors, you

431
00:19:40,050 --> 00:19:42,450
could say, here's my threshold
for an electron event.

432
00:19:42,450 --> 00:19:44,246
And you can filter
out all this noise.

433
00:19:44,246 --> 00:19:45,870
So you can say an
electron struck here,

434
00:19:45,870 --> 00:19:49,161
an electron struck over here,
an electron struck over here.

435
00:19:49,161 --> 00:19:51,930
And so you've got
better signal-to-noise,

436
00:19:51,930 --> 00:19:56,530
much tighter point spread than
you would have in a CCD camera.

437
00:19:56,530 --> 00:20:01,527
And this is what's
allowing, say, in the last--

438
00:20:01,527 --> 00:20:03,360
there was a really nice
structure in Science

439
00:20:03,360 --> 00:20:07,051
a few weeks ago of p97, which
is a AAA-ATPase with the end

440
00:20:07,051 --> 00:20:07,550
domain.

441
00:20:07,550 --> 00:20:09,133
So this sort of ties
back in, I think,

442
00:20:09,133 --> 00:20:11,435
to maybe some of
the proteasome stuff

443
00:20:11,435 --> 00:20:12,560
that you were doing before.

444
00:20:12,560 --> 00:20:17,930
P97's not a protein degradation
machine, but it's a AAA-ATPase.

445
00:20:17,930 --> 00:20:21,040
And that was at 2.3
angstroms resolution.

446
00:20:21,040 --> 00:20:23,330
And basically, what's
making this possible

447
00:20:23,330 --> 00:20:24,330
is these developments.

448
00:20:24,330 --> 00:20:26,829
AUDIENCE: Can you explain what
the fiber optic [INAUDIBLE]??

449
00:20:28,847 --> 00:20:31,180
EDWARD BRIGNOLE: Sorry, I
didn't label anything up here.

450
00:20:31,180 --> 00:20:33,470
So this is your phosphor layer.

451
00:20:33,470 --> 00:20:36,060
It's like a scintillator.

452
00:20:36,060 --> 00:20:38,711
Phosphor scintillator.

453
00:20:38,711 --> 00:20:40,585
And then down here,
you've got your detector.

454
00:20:44,330 --> 00:20:50,630
And then the fiber optics is
basically coupling the photons

455
00:20:50,630 --> 00:20:53,030
that you see here,
channeling them down

456
00:20:53,030 --> 00:20:56,366
to pixels in the detector.

457
00:20:56,366 --> 00:20:59,690
Some detectors just don't
have the fiber optics.

458
00:20:59,690 --> 00:21:02,120
They'll have a lens
of some sort here.

459
00:21:10,601 --> 00:21:11,100
All right.

460
00:21:11,100 --> 00:21:14,420
So any other questions
about EM before we will dive

461
00:21:14,420 --> 00:21:17,780
into fatty acid biosynthesis?

462
00:21:17,780 --> 00:21:19,530
OK.

463
00:21:19,530 --> 00:21:25,080
So if I show up this cartoon
with lots of different two-

464
00:21:25,080 --> 00:21:33,490
or three-letter colored short
versions for these enzymes,

465
00:21:33,490 --> 00:21:36,730
do these names, I guess they
look familiar to you now

466
00:21:36,730 --> 00:21:37,951
probably.

467
00:21:37,951 --> 00:21:38,450
OK.

468
00:21:41,410 --> 00:21:48,220
So this is the scheme for the
eukaryotic cytosolic fatty acid

469
00:21:48,220 --> 00:21:49,370
synthesis.

470
00:21:49,370 --> 00:21:53,780
There's some differences
in the bacterial system

471
00:21:53,780 --> 00:21:59,290
and the yeast system is a
little bit different also.

472
00:21:59,290 --> 00:22:01,343
But basically, to sort of--

473
00:22:01,343 --> 00:22:03,580
I don't know-- to
help me remember

474
00:22:03,580 --> 00:22:05,470
what all these enzymes
do, I like to group

475
00:22:05,470 --> 00:22:07,720
them into the enzymes that
are responsible for chain

476
00:22:07,720 --> 00:22:10,960
elongation and the enzymes
that are responsible for chain

477
00:22:10,960 --> 00:22:11,640
processing.

478
00:22:11,640 --> 00:22:15,760
So basically, the malonyl
acetyl transferase-- so

479
00:22:15,760 --> 00:22:19,150
in our fatty acid synthesis, we
have this bifunctional enzyme

480
00:22:19,150 --> 00:22:23,260
that can transfer both
malonate from malonyl-CoA

481
00:22:23,260 --> 00:22:26,420
onto the carrier protein
or acetate onto the carrier

482
00:22:26,420 --> 00:22:26,920
protein.

483
00:22:29,590 --> 00:22:31,750
In different systems,
so in bacteria,

484
00:22:31,750 --> 00:22:33,640
they've got a
malonyltransferase,

485
00:22:33,640 --> 00:22:36,489
and then they have a
specialized ketoacyl synthase

486
00:22:36,489 --> 00:22:37,780
that picks up the starter unit.

487
00:22:37,780 --> 00:22:40,031
So there's some
differences like that.

488
00:22:40,031 --> 00:22:41,530
But basically, these
are the enzymes

489
00:22:41,530 --> 00:22:43,880
that are responsible
for collecting

490
00:22:43,880 --> 00:22:48,010
the starter unit and
the elongating unit

491
00:22:48,010 --> 00:22:49,735
and joining them together.

492
00:22:49,735 --> 00:22:51,610
And then you've got
these three enzymes here,

493
00:22:51,610 --> 00:22:53,490
the ketoacyl reductase,
the dehydratase

494
00:22:53,490 --> 00:22:56,690
and the enoyl reductase that
are responsible for processing

495
00:22:56,690 --> 00:22:58,820
this beta carbon.

496
00:22:58,820 --> 00:23:01,900
So you've got the
hydroxyl, the alkene,

497
00:23:01,900 --> 00:23:03,870
and then the saturated chain.

498
00:23:03,870 --> 00:23:06,590
And then it goes around again.

499
00:23:06,590 --> 00:23:07,090
All right.

500
00:23:07,090 --> 00:23:10,120
So I mentioned that
the different organisms

501
00:23:10,120 --> 00:23:11,840
have different systems.

502
00:23:11,840 --> 00:23:17,170
So in our mitochondria and in
plants, chloroplasts and most

503
00:23:17,170 --> 00:23:18,740
bacteria have a
system like this,

504
00:23:18,740 --> 00:23:23,230
where the individual enzymes
are the dissociated players.

505
00:23:23,230 --> 00:23:28,820
In fungi, some of
these enzymes are

506
00:23:28,820 --> 00:23:31,880
joined into one of two
different polypeptides.

507
00:23:31,880 --> 00:23:36,110
And some of the names here
might look unfamiliar.

508
00:23:36,110 --> 00:23:39,500
So like this malonyl
palmitoyltransferase.

509
00:23:39,500 --> 00:23:41,930
So in this case, it's
got an acetyltransferase

510
00:23:41,930 --> 00:23:44,360
to select the starter unit
and a malonyltransferase

511
00:23:44,360 --> 00:23:45,884
to select the elongating unit.

512
00:23:45,884 --> 00:23:47,300
And then the
palmitoyltransferase,

513
00:23:47,300 --> 00:23:50,880
which transfers the
product back onto CoA.

514
00:23:50,880 --> 00:23:52,920
And this is a
bifunctional in this case.

515
00:23:52,920 --> 00:23:57,050
And then in our cytosol, we've
got this giant monster enzyme

516
00:23:57,050 --> 00:23:59,630
that's got all of the
catalytic domains fused

517
00:23:59,630 --> 00:24:01,940
into one humongous polypeptide.

518
00:24:01,940 --> 00:24:04,970
This is what attracted
me to this project

519
00:24:04,970 --> 00:24:07,370
in the first place,
just how bizarre it

520
00:24:07,370 --> 00:24:11,250
is to have all of these
enzymes all tied together.

521
00:24:11,250 --> 00:24:15,420
And then we had this one
section of the protein.

522
00:24:15,420 --> 00:24:18,650
It has homology to
methyltransferases,

523
00:24:18,650 --> 00:24:22,295
and we called it the
structural domain in the paper.

524
00:24:25,560 --> 00:24:29,150
So there is a bonus question
in the handout, which is, where

525
00:24:29,150 --> 00:24:30,440
did this domain come from?

526
00:24:30,440 --> 00:24:34,010
Why do we have
this non-functional

527
00:24:34,010 --> 00:24:38,760
methyltransferase domain
in our fatty acid synthase?

528
00:24:38,760 --> 00:24:39,750
So think about it.

529
00:24:39,750 --> 00:24:41,969
If we have time, we'll
come back to it at the end.

530
00:24:41,969 --> 00:24:43,760
Then the other cool
thing about this enzyme

531
00:24:43,760 --> 00:24:46,710
is it has to dimerize
to be active.

532
00:24:46,710 --> 00:24:52,320
And so you end up with a 550
kilodalton monster protein.

533
00:24:52,320 --> 00:24:55,440
So I mentioned that the enzyme's
responsible for elongation

534
00:24:55,440 --> 00:24:56,342
and for processing.

535
00:24:56,342 --> 00:24:57,800
And the cool thing
is when you look

536
00:24:57,800 --> 00:25:00,140
at the sequence of
the protein, you've

537
00:25:00,140 --> 00:25:03,410
got the elongation enzymes
clustered at the N-terminus,

538
00:25:03,410 --> 00:25:06,550
processing enzymes clustered
together in the middle,

539
00:25:06,550 --> 00:25:08,570
the carrier protein's
way out here at the end,

540
00:25:08,570 --> 00:25:11,190
and the thioesterase is there.

541
00:25:11,190 --> 00:25:15,830
So there was some
decades of controversy

542
00:25:15,830 --> 00:25:18,350
about how the acyl carrier
protein, which is way out here,

543
00:25:18,350 --> 00:25:20,540
would be interacting
with the enzymes, which

544
00:25:20,540 --> 00:25:22,700
are way over here at this end.

545
00:25:22,700 --> 00:25:25,340
And so a model was proposed
where the enzymes sort of

546
00:25:25,340 --> 00:25:27,740
come together in a
head-to-tail fashion.

547
00:25:27,740 --> 00:25:29,990
So you would have one going
this way and the other one

548
00:25:29,990 --> 00:25:32,140
going the other way.

549
00:25:32,140 --> 00:25:33,860
But then, that
didn't jive with some

550
00:25:33,860 --> 00:25:35,240
of the biochemical
results, which

551
00:25:35,240 --> 00:25:37,323
said that this acyl carrier
protein could interact

552
00:25:37,323 --> 00:25:40,800
with the enzymes
on its own chain.

553
00:25:40,800 --> 00:25:43,220
And so there was
this controversy

554
00:25:43,220 --> 00:25:50,810
in the field, which was
resolved in part by this crystal

555
00:25:50,810 --> 00:25:51,360
structure.

556
00:25:51,360 --> 00:25:53,844
So now we could see how
the two subunits associate

557
00:25:53,844 --> 00:25:54,510
with each other.

558
00:25:54,510 --> 00:25:57,260
So one of the chains is
just colored in white.

559
00:25:57,260 --> 00:25:59,690
The other one's got the
catalytic domains all colored

560
00:25:59,690 --> 00:26:00,290
in.

561
00:26:00,290 --> 00:26:02,360
And so you can see
the cool thing here

562
00:26:02,360 --> 00:26:06,590
is the elongation enzymes are
all clustered together down

563
00:26:06,590 --> 00:26:08,420
here like in the legs.

564
00:26:08,420 --> 00:26:11,810
And up here, in
the torso and arms,

565
00:26:11,810 --> 00:26:14,120
you've got the
processing enzymes.

566
00:26:14,120 --> 00:26:16,280
And the other cool thing
about this structure

567
00:26:16,280 --> 00:26:21,050
is if we cartoon in--

568
00:26:21,050 --> 00:26:22,550
we know the acyl
carrier protein has

569
00:26:22,550 --> 00:26:25,080
to be tethered to the C-terminus
of the ketoacyl reductase

570
00:26:25,080 --> 00:26:27,470
by a 10 amino acid linker.

571
00:26:27,470 --> 00:26:29,529
So that puts the acyl
carrier protein right here,

572
00:26:29,529 --> 00:26:31,070
and it would be
completely surrounded

573
00:26:31,070 --> 00:26:34,080
by all of the catalytic domains
that it would need to contact.

574
00:26:34,080 --> 00:26:35,410
So that's kind of cool.

575
00:26:35,410 --> 00:26:37,760
Oh, yeah and then
the thioesterase

576
00:26:37,760 --> 00:26:39,920
has a 25 residue linker.

577
00:26:39,920 --> 00:26:42,927
And so it would be
somewhere around here.

578
00:26:42,927 --> 00:26:44,510
And so basically,
all of these enzymes

579
00:26:44,510 --> 00:26:46,675
are just sitting
there in a chamber,

580
00:26:46,675 --> 00:26:48,050
and the acyl
carrier protein just

581
00:26:48,050 --> 00:26:52,600
has to bounce around to
the different things.

582
00:26:52,600 --> 00:26:57,510
So if I make a cartoon version
of the acyl carrier protein

583
00:26:57,510 --> 00:26:59,610
with its phosphopantetheine
arm docked

584
00:26:59,610 --> 00:27:01,512
into each of the
catalytic sites,

585
00:27:01,512 --> 00:27:03,720
you can see where the acyl
carrier protein would have

586
00:27:03,720 --> 00:27:06,460
to go on this reaction chamber.

587
00:27:06,460 --> 00:27:08,220
And then the same
thing would have

588
00:27:08,220 --> 00:27:11,410
to happen on the other side.

589
00:27:11,410 --> 00:27:13,550
So now I've got a
question for you.

590
00:27:13,550 --> 00:27:16,280
What happens if we make
a mutant heterodimer.

591
00:27:16,280 --> 00:27:18,280
So this is actually an
experiment that was done,

592
00:27:18,280 --> 00:27:20,529
but if we make a mutant
heterodimer where we knock out

593
00:27:20,529 --> 00:27:23,440
the ACP on this
subunit but leave

594
00:27:23,440 --> 00:27:24,860
this other subunit intact.

595
00:27:24,860 --> 00:27:27,670
So if the wild type
has 100% activity,

596
00:27:27,670 --> 00:27:30,038
how much activity
would this mutant have?

597
00:27:33,862 --> 00:27:36,896
Any guesses?

598
00:27:36,896 --> 00:27:37,770
What would you think?

599
00:27:37,770 --> 00:27:40,414
It's firing on one
of its two cylinders.

600
00:27:40,414 --> 00:27:41,320
AUDIENCE: 50%.

601
00:27:41,320 --> 00:27:43,080
EDWARD BRIGNOLE: 50%, exactly.

602
00:27:43,080 --> 00:27:44,830
So that's all good.

603
00:27:44,830 --> 00:27:46,067
That makes sense.

604
00:27:46,067 --> 00:27:47,650
What if we do another
experiment where

605
00:27:47,650 --> 00:27:52,030
we knock out the elongation
enzymes in the other reaction

606
00:27:52,030 --> 00:27:53,140
chamber?

607
00:27:53,140 --> 00:27:54,070
Now what do you think?

608
00:27:58,635 --> 00:28:00,260
AUDIENCE: Expect it
wouldn't be active.

609
00:28:00,260 --> 00:28:02,718
EDWARD BRIGNOLE: You'd expect
it wouldn't be active at all.

610
00:28:02,718 --> 00:28:05,570
But the experimental results
show that it had about 25%

611
00:28:05,570 --> 00:28:06,980
activity.

612
00:28:06,980 --> 00:28:09,230
So the only way
that could happen

613
00:28:09,230 --> 00:28:12,680
is if this acyl carrier protein
can elongate with the enzymes

614
00:28:12,680 --> 00:28:14,180
from the opposite chain.

615
00:28:14,180 --> 00:28:15,530
Right?

616
00:28:15,530 --> 00:28:18,260
So that looks like
a pretty long reach,

617
00:28:18,260 --> 00:28:20,930
but let's figure out
how far that would be.

618
00:28:20,930 --> 00:28:24,080
So a 10 residue linker to
the acyl carrier protein

619
00:28:24,080 --> 00:28:25,580
would be about 35 angstroms.

620
00:28:25,580 --> 00:28:28,340
The acyl carrier protein
itself is about 23.

621
00:28:28,340 --> 00:28:30,890
Then you have the
phosphopantetheine arm,

622
00:28:30,890 --> 00:28:33,440
which would be these
black things coming off

623
00:28:33,440 --> 00:28:34,820
of our acyl carrier protein.

624
00:28:34,820 --> 00:28:39,190
So if we draw that to scale
from the C-terminus of the keto

625
00:28:39,190 --> 00:28:42,780
reductase to the end
of the red sphere,

626
00:28:42,780 --> 00:28:44,210
it would be about 60 angstroms.

627
00:28:44,210 --> 00:28:47,615
So if we draw how
big that would be,

628
00:28:47,615 --> 00:28:50,070
that's this gray
sphere right here.

629
00:28:50,070 --> 00:28:52,700
So this is how far the acyl
carrier protein can reach,

630
00:28:52,700 --> 00:28:55,640
and you can see these
are clearly out of range.

631
00:28:55,640 --> 00:28:57,590
And actually, even
the elongation enzymes

632
00:28:57,590 --> 00:29:01,160
in its own side are also
sort of at the limit

633
00:29:01,160 --> 00:29:03,830
of what the acyl carrier
protein can reach to.

634
00:29:03,830 --> 00:29:07,079
So it's hard to imagine
what would happen,

635
00:29:07,079 --> 00:29:09,620
but you would need to have some
sort of conformational change

636
00:29:09,620 --> 00:29:11,750
to make these things happen.

637
00:29:11,750 --> 00:29:13,790
And I'll point out one
other difficulty, which

638
00:29:13,790 --> 00:29:16,340
is the access to
the enoyl reductase

639
00:29:16,340 --> 00:29:19,430
and the dehydratase are
sandwiched in the space

640
00:29:19,430 --> 00:29:20,950
between them there.

641
00:29:20,950 --> 00:29:24,740
And so you'd need to have
some other separation

642
00:29:24,740 --> 00:29:27,140
of these domains, possibly,
to get the acyl carrier

643
00:29:27,140 --> 00:29:29,190
protein in there.

644
00:29:29,190 --> 00:29:34,290
So we wanted to look at this EM.

645
00:29:34,290 --> 00:29:36,290
Do you know why that would
seem like a good idea

646
00:29:36,290 --> 00:29:39,770
based on what we talked
about with EM so far?

647
00:29:39,770 --> 00:29:41,690
Does fatty acid
synthase seem like it

648
00:29:41,690 --> 00:29:44,574
would be a good target for EM?

649
00:29:44,574 --> 00:29:46,740
I mean, there was already
a crystal structure of it.

650
00:29:46,740 --> 00:29:50,180
So should we have tried
crystallography, say,

651
00:29:50,180 --> 00:29:52,535
to answer questions about
conformational changes?

652
00:29:57,140 --> 00:29:59,300
AUDIENCE: You might not
be able to crystallize it

653
00:29:59,300 --> 00:30:00,756
in the conformation you wanted?

654
00:30:00,756 --> 00:30:02,130
EDWARD BRIGNOLE:
Yeah, as it was,

655
00:30:02,130 --> 00:30:04,160
that was difficult
molecule to crystallize.

656
00:30:04,160 --> 00:30:07,980
There were crystals of
it from back in the maybe

657
00:30:07,980 --> 00:30:12,840
'70s, '80s, but it wasn't
until mid-2000s that they had

658
00:30:12,840 --> 00:30:13,950
actually gotten--

659
00:30:13,950 --> 00:30:16,670
they solved it initially at, I
think, six or seven angstroms.

660
00:30:16,670 --> 00:30:19,380
And then this structure
was, I think, also not

661
00:30:19,380 --> 00:30:21,630
the highest resolution,
somewhere in the three to four

662
00:30:21,630 --> 00:30:23,577
range.

663
00:30:23,577 --> 00:30:26,160
So yeah, you would have to find
ways to trap the conformations

664
00:30:26,160 --> 00:30:28,680
that you want, lock it
in, and cross your fingers

665
00:30:28,680 --> 00:30:31,230
to get crystals.

666
00:30:31,230 --> 00:30:33,260
Why look at it by
electron microscopy?

667
00:30:36,220 --> 00:30:39,490
Is it big enough?

668
00:30:39,490 --> 00:30:43,930
It's 550 kilodaltons that you
can see individual molecules.

669
00:30:43,930 --> 00:30:46,979
Possibly we could even see
them in different states,

670
00:30:46,979 --> 00:30:49,270
and we might even be able to
perturb those states if we

671
00:30:49,270 --> 00:30:51,790
threw in some substrates.

672
00:30:51,790 --> 00:30:55,240
Then we had a whole
panel of mutants

673
00:30:55,240 --> 00:30:57,509
that our collaborator had meant.

674
00:30:57,509 --> 00:30:59,800
The experiments that I had
described about knocking out

675
00:30:59,800 --> 00:31:02,290
the ACP in one chain
versus the elongation

676
00:31:02,290 --> 00:31:05,890
enzymes in the other, there
was a whole battery of mutants

677
00:31:05,890 --> 00:31:07,540
that we had available to us.

678
00:31:07,540 --> 00:31:08,720
All right.

679
00:31:08,720 --> 00:31:11,402
So to do the
electron microscopy,

680
00:31:11,402 --> 00:31:13,360
we need to put our protein
on something that we

681
00:31:13,360 --> 00:31:14,610
can stick into the microscope.

682
00:31:14,610 --> 00:31:18,760
And typically,
that's a metal mesh

683
00:31:18,760 --> 00:31:22,000
with a cart that's supporting
a thin carbon film.

684
00:31:22,000 --> 00:31:25,280
And then we stick the protein
onto the thin carbon film.

685
00:31:25,280 --> 00:31:27,040
So this is about
three millimeters

686
00:31:27,040 --> 00:31:28,210
across, this little grid.

687
00:31:28,210 --> 00:31:30,160
You can put about five
microliters on it.

688
00:31:30,160 --> 00:31:33,100
And then to get a good
dispersion of particles

689
00:31:33,100 --> 00:31:36,400
on the grid, you need about
15 nanograms per microliter.

690
00:31:36,400 --> 00:31:39,790
If you go too much above that,
you get protein everywhere,

691
00:31:39,790 --> 00:31:41,960
and you can't pick one
particle from another.

692
00:31:41,960 --> 00:31:45,610
And if you go much
below that, then you

693
00:31:45,610 --> 00:31:49,000
have to collect lots and lots of
images to get a few particles.

694
00:31:49,000 --> 00:31:51,940
So this is sort of the
sweet spot, in the 15 to 20

695
00:31:51,940 --> 00:31:54,250
nanogram per microliter range.

696
00:31:54,250 --> 00:31:59,330
This is one limitation for EM,
the concentration dependence.

697
00:31:59,330 --> 00:32:04,660
So if you have a molecule that
falls apart, it has a high Kd

698
00:32:04,660 --> 00:32:06,640
and it falls apart at
these concentrations,

699
00:32:06,640 --> 00:32:10,030
that could be difficult to
work with, for instance.

700
00:32:10,030 --> 00:32:11,262
All right.

701
00:32:11,262 --> 00:32:13,720
So there's a couple of different
ways to prepare specimens.

702
00:32:13,720 --> 00:32:15,969
I think we already talked
about staining the specimens

703
00:32:15,969 --> 00:32:17,820
or cryogenically
preserving them.

704
00:32:17,820 --> 00:32:22,720
So the way that would look
like for a stain experiment is,

705
00:32:22,720 --> 00:32:24,430
you've got your
thin carbon film,

706
00:32:24,430 --> 00:32:27,250
you put your drop with your
protein molecules on it,

707
00:32:27,250 --> 00:32:29,830
you blot off the
excess solution,

708
00:32:29,830 --> 00:32:31,900
replace it with a heavy
metal salt solution--

709
00:32:31,900 --> 00:32:34,590
typically a uranium salt--

710
00:32:34,590 --> 00:32:36,070
and then you let it air dry.

711
00:32:36,070 --> 00:32:41,404
And the specimen is then
embedded in this heavy metal.

712
00:32:41,404 --> 00:32:42,820
And that's why we
call it negative

713
00:32:42,820 --> 00:32:48,830
stain because what
we're imaging is,

714
00:32:48,830 --> 00:32:51,060
you've got your
protein molecule,

715
00:32:51,060 --> 00:32:53,580
and it's embedded in
this dense stain layer.

716
00:32:59,220 --> 00:33:02,820
What's scattering the
electrons most strongly

717
00:33:02,820 --> 00:33:04,810
is the material
around your specimen.

718
00:33:04,810 --> 00:33:09,810
And so you're imaging where
your protein isn't, basically.

719
00:33:09,810 --> 00:33:12,240
Or the stain excluded area
is what you're imaging.

720
00:33:12,240 --> 00:33:15,870
So what you have, in this
case, is a dark background,

721
00:33:15,870 --> 00:33:18,500
and your particles look light.

722
00:33:18,500 --> 00:33:21,060
The other way to
prepare specimens

723
00:33:21,060 --> 00:33:23,170
is to cryogenically
preserve them.

724
00:33:23,170 --> 00:33:24,810
So the first part
starts out the same.

725
00:33:24,810 --> 00:33:26,919
You would put your
proteins on the grid.

726
00:33:26,919 --> 00:33:28,710
And sometimes, you
could have a grid that's

727
00:33:28,710 --> 00:33:30,660
got little perforations
in the carbon,

728
00:33:30,660 --> 00:33:33,960
so you actually would have
your protein suspended

729
00:33:33,960 --> 00:33:35,640
in these perforations
when you blot it.

730
00:33:35,640 --> 00:33:37,870
And then you plunge
it into liquid ethane

731
00:33:37,870 --> 00:33:42,400
that's cooled to just about to
liquid nitrogen temperatures.

732
00:33:42,400 --> 00:33:45,099
Here is a picture of the dewar
with the liquid nitrogen.

733
00:33:45,099 --> 00:33:46,890
And then there's a
little cup in the middle

734
00:33:46,890 --> 00:33:48,984
with the liquid ethane.

735
00:33:48,984 --> 00:33:50,400
So why not just
plunge it directly

736
00:33:50,400 --> 00:33:51,441
into the liquid nitrogen?

737
00:33:54,491 --> 00:33:55,690
Does anybody know?

738
00:33:55,690 --> 00:33:59,157
Does anybody do
rapid freeze quench

739
00:33:59,157 --> 00:34:01,240
for any of your experiments
or anything like that?

740
00:34:04,360 --> 00:34:07,360
So have you ever messed
around with liquid nitrogen

741
00:34:07,360 --> 00:34:10,090
that any splashed onto you?

742
00:34:10,090 --> 00:34:11,690
Did you get burnt?

743
00:34:11,690 --> 00:34:12,190
No.

744
00:34:12,190 --> 00:34:16,929
So the reason is liquid nitrogen
has a lower heat capacity,

745
00:34:16,929 --> 00:34:19,750
so if it touches you,
basically, there's

746
00:34:19,750 --> 00:34:23,440
a layer of gas between
the liquid and your hand

747
00:34:23,440 --> 00:34:25,980
or whatever it spilled on.

748
00:34:25,980 --> 00:34:29,889
But with liquid ethane,
the heat transfer--

749
00:34:29,889 --> 00:34:32,679
basically, this grid
will go in there,

750
00:34:32,679 --> 00:34:36,639
and it'll freeze so fast that
ice doesn't have a chance

751
00:34:36,639 --> 00:34:38,230
to form crystalline ice.

752
00:34:38,230 --> 00:34:42,730
So basically, everything is,
on a microsecond scale, frozen.

753
00:34:45,690 --> 00:34:48,840
So now you've got
this amorphous ice

754
00:34:48,840 --> 00:34:52,830
with your protein
embedded in it.

755
00:34:52,830 --> 00:34:55,889
Can you think of some
advantages or disadvantages?

756
00:34:55,889 --> 00:34:57,700
If this gives you
something preserved

757
00:34:57,700 --> 00:34:59,692
and it's happy in
its buffer, why

758
00:34:59,692 --> 00:35:00,900
wouldn't you always use that?

759
00:35:00,900 --> 00:35:04,550
Why would you use stain?

760
00:35:04,550 --> 00:35:07,660
Can you think of
some advantages?

761
00:35:07,660 --> 00:35:09,160
Maybe the obvious
thing, why don't I

762
00:35:09,160 --> 00:35:10,591
ask you for some disadvantages.

763
00:35:10,591 --> 00:35:12,090
Why would you not
want to use stain?

764
00:35:16,018 --> 00:35:19,327
AUDIENCE: The stain used could
possibly disrupt your specimen.

765
00:35:19,327 --> 00:35:21,160
EDWARD BRIGNOLE: Yeah,
and that does happen.

766
00:35:23,800 --> 00:35:26,410
Sometimes people have to play
around with different stains.

767
00:35:26,410 --> 00:35:29,830
The uranium salt stains, the
uranyl acetate, for instance,

768
00:35:29,830 --> 00:35:31,040
is a low pH.

769
00:35:31,040 --> 00:35:33,060
And if you try to pH
it, it crashes out.

770
00:35:33,060 --> 00:35:39,070
So if your protein isn't
happy in that low pH stain,

771
00:35:39,070 --> 00:35:40,766
that could be a problem.

772
00:35:40,766 --> 00:35:43,270
Also the stain layer is
dried, and so your specimen

773
00:35:43,270 --> 00:35:45,400
is dehydrated and
dried out here.

774
00:35:45,400 --> 00:35:46,330
And typically, that--

775
00:35:49,060 --> 00:35:50,710
I drew my specimen like this.

776
00:35:50,710 --> 00:35:55,330
But when it dries out, it
flattens out like this.

777
00:35:58,520 --> 00:36:00,860
So that's a disadvantage.

778
00:36:00,860 --> 00:36:03,440
What about contrast?

779
00:36:03,440 --> 00:36:09,365
So if this is my amorphous ice,
my water layer with my protein,

780
00:36:09,365 --> 00:36:11,740
do you know what the difference
in the density of protein

781
00:36:11,740 --> 00:36:14,080
versus an aqueous buffer is?

782
00:36:17,712 --> 00:36:20,360
They're pretty closely
matched, actually.

783
00:36:20,360 --> 00:36:22,610
Protein's like 1.2 or
something like that.

784
00:36:22,610 --> 00:36:26,360
So basically, you have
pretty weak contrast

785
00:36:26,360 --> 00:36:28,910
in a frozen hydrated
specimen because here, you're

786
00:36:28,910 --> 00:36:30,770
looking at the
difference in density

787
00:36:30,770 --> 00:36:32,600
of your protein versus
the buffer around it

788
00:36:32,600 --> 00:36:34,391
whereas here, you're
imaging the difference

789
00:36:34,391 --> 00:36:38,220
between the density of, say,
your protein and uranium.

790
00:36:38,220 --> 00:36:41,042
So you get a lot
better signal here.

791
00:36:41,042 --> 00:36:42,750
But you've got some
specimen distortions.

792
00:36:42,750 --> 00:36:46,680
And so we basically
just went through these.

793
00:36:46,680 --> 00:36:49,010
There's one other
advantage I'll mention

794
00:36:49,010 --> 00:36:52,040
to sticking your protein onto
a carbon surface as opposed

795
00:36:52,040 --> 00:36:56,852
to freezing your
protein in a hole.

796
00:36:56,852 --> 00:36:58,310
And that is that
most proteins tend

797
00:36:58,310 --> 00:36:59,810
to have a preferred orientation.

798
00:36:59,810 --> 00:37:02,360
Many do, and in the case
of fatty acid synthase,

799
00:37:02,360 --> 00:37:03,330
it's sort of this.

800
00:37:03,330 --> 00:37:05,872
It looks like a headless person
that's got arms and legs.

801
00:37:05,872 --> 00:37:08,080
It'll very rarely hit the
grid and stand straight up.

802
00:37:08,080 --> 00:37:10,360
It usually falls
back onto its back.

803
00:37:10,360 --> 00:37:13,460
And so in some
circumstances, that

804
00:37:13,460 --> 00:37:15,580
could be an advantage,
and other circumstances

805
00:37:15,580 --> 00:37:18,080
you would actually want to have
many different views to make

806
00:37:18,080 --> 00:37:19,610
a 3D structure.

807
00:37:19,610 --> 00:37:23,940
So I listed that both as an
advantage and a disadvantage,

808
00:37:23,940 --> 00:37:25,390
the preferred orientation.

809
00:37:25,390 --> 00:37:26,000
Depends.

810
00:37:26,000 --> 00:37:27,881
You could use it
to your advantage.

811
00:37:27,881 --> 00:37:29,630
In other cases it would
be a disadvantage.

812
00:37:29,630 --> 00:37:30,830
All right.

813
00:37:30,830 --> 00:37:33,560
So you said you used
an FEI microscope.

814
00:37:33,560 --> 00:37:35,085
It might have looked
like this one.

815
00:37:35,085 --> 00:37:35,710
AUDIENCE: Yeah.

816
00:37:35,710 --> 00:37:36,544
[INTERPOSING VOICES]

817
00:37:36,544 --> 00:37:37,376
EDWARD BRIGNOLE: OK.

818
00:37:37,376 --> 00:37:38,330
Yeah, this is an F20.

819
00:37:38,330 --> 00:37:40,820
This is the microscope that
all the images in the paper

820
00:37:40,820 --> 00:37:42,910
were collected on.

821
00:37:42,910 --> 00:37:44,990
So there is a specimen
port on the side.

822
00:37:44,990 --> 00:37:46,850
The electron source
is up here at the top.

823
00:37:46,850 --> 00:37:49,910
The column with the lenses
and apertures in it is here.

824
00:37:49,910 --> 00:37:52,004
There's a phosphorus
screen here that you

825
00:37:52,004 --> 00:37:53,420
can look at through
the binoculars

826
00:37:53,420 --> 00:37:54,929
to see what's going on.

827
00:37:54,929 --> 00:37:57,470
There's the knobs that you can
use to control the microscope,

828
00:37:57,470 --> 00:37:59,540
focus, move the stage around.

829
00:37:59,540 --> 00:38:02,540
And then the camera is
right here below the column,

830
00:38:02,540 --> 00:38:04,490
right where you can
knock your knees into it

831
00:38:04,490 --> 00:38:08,180
when you look in here.

832
00:38:08,180 --> 00:38:10,771
Yeah, I mean, you put a half
a million dollar detector

833
00:38:10,771 --> 00:38:11,270
on there.

834
00:38:11,270 --> 00:38:14,710
And you can knock
your knees into it.

835
00:38:14,710 --> 00:38:16,460
Actually, the newer
microscopes these days

836
00:38:16,460 --> 00:38:18,830
actually look more like
giant refrigerators.

837
00:38:18,830 --> 00:38:21,749
And basically, all
of this is housed

838
00:38:21,749 --> 00:38:24,290
in this environmental chamber,
and you operate the microscope

839
00:38:24,290 --> 00:38:25,615
from the room next door.

840
00:38:29,360 --> 00:38:32,520
So we put the grid
in the microscope.

841
00:38:32,520 --> 00:38:35,000
At low mag, you can
get an image like this.

842
00:38:35,000 --> 00:38:38,570
Little higher, just zooming in
on one of these squares here,

843
00:38:38,570 --> 00:38:39,880
you can get an image like this.

844
00:38:39,880 --> 00:38:41,630
This is negative stain
specimen so there's

845
00:38:41,630 --> 00:38:44,010
little chunks of stain around.

846
00:38:44,010 --> 00:38:47,550
If you ever happened to do some
negative stain experiments,

847
00:38:47,550 --> 00:38:50,210
I usually like to
look for areas that

848
00:38:50,210 --> 00:38:52,240
have this smudgy appearance.

849
00:38:52,240 --> 00:38:53,967
It looks like little
pencil lead shavings

850
00:38:53,967 --> 00:38:55,550
that somebody wiped
their hand across.

851
00:38:55,550 --> 00:38:57,580
That's usually a good sign.

852
00:38:57,580 --> 00:38:59,720
And then if you zoom
in another tenfold,

853
00:38:59,720 --> 00:39:01,680
you can get an image like this.

854
00:39:01,680 --> 00:39:03,410
And if you look
carefully at it, there's

855
00:39:03,410 --> 00:39:07,540
all the individual 550
kilodalton fatty acid synthase

856
00:39:07,540 --> 00:39:08,120
molecules.

857
00:39:08,120 --> 00:39:15,280
So how do you get any
information out of that?

858
00:39:15,280 --> 00:39:15,990
Any ideas?

859
00:39:20,960 --> 00:39:22,994
You can pick out the
individual molecules here.

860
00:39:22,994 --> 00:39:24,410
If you squint at
it, can you maybe

861
00:39:24,410 --> 00:39:27,620
make out the legs and arms,
the processing portion,

862
00:39:27,620 --> 00:39:31,150
and the elongation portion?

863
00:39:31,150 --> 00:39:33,000
Maybe?

864
00:39:33,000 --> 00:39:34,370
OK, it's tough.

865
00:39:34,370 --> 00:39:36,800
Does anybody here
do spectroscopy?

866
00:39:36,800 --> 00:39:37,520
AUDIENCE: No.

867
00:39:37,520 --> 00:39:38,353
EDWARD BRIGNOLE: No.

868
00:39:41,330 --> 00:39:44,630
So based on what you know,
electron microscope images

869
00:39:44,630 --> 00:39:48,740
can have potentially high
resolution information in them.

870
00:39:48,740 --> 00:39:53,060
But you're limited in dose
you can apply to the specimen

871
00:39:53,060 --> 00:39:56,250
before radiation damage
becomes a problem.

872
00:39:56,250 --> 00:39:59,160
So what we have is a
signal-to-noise problem here.

873
00:39:59,160 --> 00:40:01,790
You've got high resolution
signal buried in lots of noise.

874
00:40:04,670 --> 00:40:09,170
It's like having a low
exposure image of something.

875
00:40:09,170 --> 00:40:12,089
What could you do to
boost your signal?

876
00:40:12,089 --> 00:40:13,880
If you're going to take
a picture at night,

877
00:40:13,880 --> 00:40:16,744
what would you do?

878
00:40:16,744 --> 00:40:18,410
You need a really,
really long exposure.

879
00:40:18,410 --> 00:40:19,460
Right?

880
00:40:19,460 --> 00:40:22,011
But you can't take a really,
really long exposure.

881
00:40:22,011 --> 00:40:23,760
So what would be a
different way to do it?

882
00:40:26,910 --> 00:40:29,235
Say like, in the
case of spectroscopy,

883
00:40:29,235 --> 00:40:31,110
if you had a sample
that's damaged every time

884
00:40:31,110 --> 00:40:34,100
you stuck the
cuvette in the area,

885
00:40:34,100 --> 00:40:36,996
but you could say, take a
cuvette and take a spectra,

886
00:40:36,996 --> 00:40:39,120
take another one, take a
spectra, take another one,

887
00:40:39,120 --> 00:40:42,270
take a spectra, and you can
average lots of them together,

888
00:40:42,270 --> 00:40:44,214
that would boost
your signal-to-noise.

889
00:40:44,214 --> 00:40:45,630
So that's what we
have to do here.

890
00:40:45,630 --> 00:40:49,710
We have to extract all these
particles out and find a way

891
00:40:49,710 --> 00:40:51,040
to average them together.

892
00:40:51,040 --> 00:40:53,820
So if we put soccer
players on a EM grid--

893
00:40:53,820 --> 00:40:56,790
if any of you are soccer fans--

894
00:40:56,790 --> 00:40:59,170
and you collect
an image of them.

895
00:40:59,170 --> 00:41:02,000
You get this noisy
image like this.

896
00:41:02,000 --> 00:41:04,630
In the computer, you can go
through and pick the particles

897
00:41:04,630 --> 00:41:05,130
out.

898
00:41:05,130 --> 00:41:07,920
And the computer can do its
best to line them up for you.

899
00:41:07,920 --> 00:41:09,375
And if it does a
good job, and you

900
00:41:09,375 --> 00:41:12,000
get lots and lots of particles,
when you average them together,

901
00:41:12,000 --> 00:41:17,020
you get your high
resolution signal out.

902
00:41:17,020 --> 00:41:19,470
So that's all fine and
good, but not every protein

903
00:41:19,470 --> 00:41:21,600
is going to land in exactly
the same orientation.

904
00:41:21,600 --> 00:41:23,016
And in the case
of soccer players,

905
00:41:23,016 --> 00:41:25,712
you probably would have a hard
time finding soccer players

906
00:41:25,712 --> 00:41:27,420
that are in exactly
the same conformation

907
00:41:27,420 --> 00:41:29,000
every time you image them.

908
00:41:29,000 --> 00:41:32,970
So in this case, a
soccer player might

909
00:41:32,970 --> 00:41:35,460
prefer to kick with his
right foot or left foot

910
00:41:35,460 --> 00:41:37,817
or might have his right
arm or left arm up or down.

911
00:41:37,817 --> 00:41:39,900
These are just a couple
of different conformations

912
00:41:39,900 --> 00:41:41,108
maybe that you would observe.

913
00:41:41,108 --> 00:41:42,894
So now what do you do?

914
00:41:42,894 --> 00:41:45,060
You've got these averaged
together, and you're like,

915
00:41:45,060 --> 00:41:45,900
I got an insect.

916
00:41:51,600 --> 00:41:55,650
Does anybody here-- have
you looked at, say--

917
00:41:55,650 --> 00:41:57,090
you could do this
by spectroscopy.

918
00:41:57,090 --> 00:41:59,131
But it sounds like nobody
here does spectroscopy.

919
00:41:59,131 --> 00:42:01,740
So you've got different
sorts of things

920
00:42:01,740 --> 00:42:04,110
that you want to
categorize, basically.

921
00:42:04,110 --> 00:42:07,740
So say, sequence
alignments, that

922
00:42:07,740 --> 00:42:10,230
would be analogous to
this, where you've got

923
00:42:10,230 --> 00:42:11,691
sequences that you've lined up.

924
00:42:11,691 --> 00:42:13,440
Here we've got images
that we've lined up.

925
00:42:13,440 --> 00:42:14,670
And then what would you do?

926
00:42:14,670 --> 00:42:19,110
You'd look through columns
of residues or the computer

927
00:42:19,110 --> 00:42:22,440
would do this for you and say,
this cluster of sequences all

928
00:42:22,440 --> 00:42:25,320
have these particular residues.

929
00:42:25,320 --> 00:42:27,121
So I'm going to put
them into one bin.

930
00:42:27,121 --> 00:42:28,620
And these have a
different sequence,

931
00:42:28,620 --> 00:42:30,720
and I'm going to put those
into a different bin.

932
00:42:30,720 --> 00:42:32,670
The computer can do the same
thing in this cage, basically.

933
00:42:32,670 --> 00:42:34,670
It'll look at these images
and say, some of them

934
00:42:34,670 --> 00:42:37,350
have a density here, and some
of them have a density there

935
00:42:37,350 --> 00:42:41,874
and split them up based on
differences in the intensities

936
00:42:41,874 --> 00:42:42,540
of these pixels.

937
00:42:45,110 --> 00:42:47,980
If this is a dataset of 100
images, then you split it.

938
00:42:47,980 --> 00:42:49,890
Now you've got 50 and 50.

939
00:42:49,890 --> 00:42:52,890
You might have 25, 25, 25,
25 if everything's evenly

940
00:42:52,890 --> 00:42:54,187
distributed.

941
00:42:54,187 --> 00:42:56,520
And the one thing you'll
notice as you split things down

942
00:42:56,520 --> 00:42:59,110
further, you're averaging fewer
and fewer particles together.

943
00:42:59,110 --> 00:43:02,330
And so your signal-to-noise
is getting worse and worse.

944
00:43:02,330 --> 00:43:04,210
So you can split these down.

945
00:43:04,210 --> 00:43:07,486
And the way I typically do
this is a little bit empirical,

946
00:43:07,486 --> 00:43:09,360
but I'll split it and
then split it some more

947
00:43:09,360 --> 00:43:11,760
and split it some more and
look till I get to a point

948
00:43:11,760 --> 00:43:14,700
where I'm not
seeing anything new.

949
00:43:14,700 --> 00:43:16,500
Because if you split
this image more,

950
00:43:16,500 --> 00:43:18,750
it's basically going to be
split on the basis of noise

951
00:43:18,750 --> 00:43:21,224
because there's no other
conformational change.

952
00:43:21,224 --> 00:43:22,890
The same thing would
go for orientation.

953
00:43:22,890 --> 00:43:23,940
If you put this
on the grid and it

954
00:43:23,940 --> 00:43:25,710
landed in three
different orientations,

955
00:43:25,710 --> 00:43:27,085
you would want to
separate things

956
00:43:27,085 --> 00:43:30,039
out using the same strategy.

957
00:43:30,039 --> 00:43:31,830
All right, so this gets
us to the averages,

958
00:43:31,830 --> 00:43:34,350
like the averages that you
see in the fatty acid synthase

959
00:43:34,350 --> 00:43:35,880
paper.

960
00:43:35,880 --> 00:43:38,430
There are also 3D
structures in the paper.

961
00:43:38,430 --> 00:43:40,860
So how do you go
from information

962
00:43:40,860 --> 00:43:44,140
like this to a 3D structure?

963
00:43:44,140 --> 00:43:45,640
So I mentioned one
way is if you've

964
00:43:45,640 --> 00:43:47,380
got lots of different
orientations of the molecule

965
00:43:47,380 --> 00:43:49,840
on the grid and you've got
each one of these averages

966
00:43:49,840 --> 00:43:53,107
is a different view,
you can use the computer

967
00:43:53,107 --> 00:43:54,940
to try to put those
different views together

968
00:43:54,940 --> 00:43:56,320
to come up with a 3D structure.

969
00:43:56,320 --> 00:43:58,111
And there's lots of
ways to get that wrong.

970
00:44:00,702 --> 00:44:02,660
In this case, we've got
a preferred orientation

971
00:44:02,660 --> 00:44:04,284
where they're all
lying on their backs.

972
00:44:04,284 --> 00:44:06,214
We don't have lots
of different views.

973
00:44:06,214 --> 00:44:08,162
So what would you do instead?

974
00:44:11,680 --> 00:44:12,180
Yeah?

975
00:44:12,180 --> 00:44:13,799
AUDIENCE: Get it from the sides?

976
00:44:13,799 --> 00:44:15,090
EDWARD BRIGNOLE: Yeah, exactly.

977
00:44:15,090 --> 00:44:20,460
So that's the thing to do.

978
00:44:20,460 --> 00:44:24,180
So now you've got this
stereo view of your molecule

979
00:44:24,180 --> 00:44:27,810
where you've got a tilted
view and an untilted view.

980
00:44:27,810 --> 00:44:29,690
An extreme example
of this would be

981
00:44:29,690 --> 00:44:32,310
if every particle-- let's say
if you're looking at cells,

982
00:44:32,310 --> 00:44:33,914
no two cells are the same.

983
00:44:33,914 --> 00:44:35,580
You couldn't do these
averaging methods,

984
00:44:35,580 --> 00:44:38,121
but to get a 3D reconstruction
of a cell, what you would have

985
00:44:38,121 --> 00:44:40,180
to do is take the stage
and tilt it by a degree,

986
00:44:40,180 --> 00:44:42,162
tilt it by a degree,
tilt it by a degree,

987
00:44:42,162 --> 00:44:44,370
go up as far as you can one
way, and then do it again

988
00:44:44,370 --> 00:44:44,953
the other way.

989
00:44:44,953 --> 00:44:49,120
So you'd have up to maybe
plus or minus 70 degrees.

990
00:44:49,120 --> 00:44:52,530
And that would be equivalent to
the way a CAT scan or something

991
00:44:52,530 --> 00:44:56,482
might be done, where you've
got images of your broken leg

992
00:44:56,482 --> 00:44:58,440
or something like that
from all the way around.

993
00:44:58,440 --> 00:45:00,523
And then you can have the
3D reconstruction of it.

994
00:45:03,130 --> 00:45:03,780
Right.

995
00:45:03,780 --> 00:45:06,060
So we've got these two
images of our specimen.

996
00:45:06,060 --> 00:45:09,050
They're related to each other
by some tilt that you know.

997
00:45:09,050 --> 00:45:11,430
If you take out these
particles from this image

998
00:45:11,430 --> 00:45:13,320
and you line them
up, that tells you

999
00:45:13,320 --> 00:45:15,720
what view you've
got of them in this.

1000
00:45:15,720 --> 00:45:19,840
So take, for instance,
this molecule here.

1001
00:45:19,840 --> 00:45:22,050
If you have to rotate
this 90 degrees clockwise,

1002
00:45:22,050 --> 00:45:23,549
that tells you
you're looking at him

1003
00:45:23,549 --> 00:45:26,580
in the tilted view with
his feet up in the air.

1004
00:45:26,580 --> 00:45:29,112
And this one here had to go
90 degrees counterclockwise.

1005
00:45:29,112 --> 00:45:30,570
That means that
you're looking down

1006
00:45:30,570 --> 00:45:33,740
on him in this tilted
view with his head up.

1007
00:45:33,740 --> 00:45:36,030
And so you can take the
alignment information

1008
00:45:36,030 --> 00:45:39,660
from this image and apply
that as a projection

1009
00:45:39,660 --> 00:45:43,170
parameter for these images.

1010
00:45:43,170 --> 00:45:46,050
And so you could take now these
tilted views of these soccer

1011
00:45:46,050 --> 00:45:48,230
players, and you know
which view they are.

1012
00:45:48,230 --> 00:45:51,850
And you can come up with the
3D reconstruction that way.

1013
00:45:51,850 --> 00:45:57,060
So this is actually
a fairly old method.

1014
00:45:57,060 --> 00:45:59,910
I think it's still widely
used and very elegant

1015
00:45:59,910 --> 00:46:02,659
because it's basically just
two images of the same thing.

1016
00:46:02,659 --> 00:46:04,200
And then you can
get a reconstruction

1017
00:46:04,200 --> 00:46:07,560
that's pretty easy to get out.

1018
00:46:07,560 --> 00:46:13,534
There is one disadvantage
to this approach, one

1019
00:46:13,534 --> 00:46:15,450
major disadvantage, which
is that you can only

1020
00:46:15,450 --> 00:46:17,370
tilt the stage so far.

1021
00:46:17,370 --> 00:46:20,660
So if you could tilt the
stage up to 90 degrees,

1022
00:46:20,660 --> 00:46:23,460
then you would have views
exactly all the way around.

1023
00:46:23,460 --> 00:46:26,430
And you could have
a reconstruction

1024
00:46:26,430 --> 00:46:29,040
that's fully complete.

1025
00:46:29,040 --> 00:46:31,180
In this case, you can
only tilt to 70 degrees,

1026
00:46:31,180 --> 00:46:35,300
and so you've got a
missing cone of information

1027
00:46:35,300 --> 00:46:38,492
in the reconstruction.

1028
00:46:38,492 --> 00:46:39,950
And so basically
what that means is

1029
00:46:39,950 --> 00:46:43,020
you've got better resolution
in x and y than you do in z.

1030
00:46:43,020 --> 00:46:43,580
Yeah, sure?

1031
00:46:43,580 --> 00:46:49,169
AUDIENCE: Is there some graphene
packet thing that came out

1032
00:46:49,169 --> 00:46:53,640
of the [INAUDIBLE] lab where
it's this packet filled with

1033
00:46:53,640 --> 00:46:59,026
solution that you
shoot your EM at the--

1034
00:46:59,026 --> 00:47:02,170
EDWARD BRIGNOLE: Yeah, to
keep your protein hydrated,

1035
00:47:02,170 --> 00:47:03,040
basically.

1036
00:47:03,040 --> 00:47:05,830
So you encapsulate
it in some sort

1037
00:47:05,830 --> 00:47:07,420
of graphene tube or something.

1038
00:47:07,420 --> 00:47:10,432
AUDIENCE: [INAUDIBLE]
exactly [INAUDIBLE]

1039
00:47:10,432 --> 00:47:11,890
EDWARD BRIGNOLE:
I vaguely remember

1040
00:47:11,890 --> 00:47:14,512
seeing something like that.

1041
00:47:14,512 --> 00:47:16,720
I think there are groups
working on things like that,

1042
00:47:16,720 --> 00:47:19,262
but it's not widely
adopted or used yet.

1043
00:47:19,262 --> 00:47:21,220
But yeah, there are some
pretty exciting things

1044
00:47:21,220 --> 00:47:23,890
like that that might allow
you to directly image

1045
00:47:23,890 --> 00:47:26,500
your molecule while it's
tumbling in solution, isolated

1046
00:47:26,500 --> 00:47:28,420
from the vacuum
on the microscope.

1047
00:47:28,420 --> 00:47:30,820
AUDIENCE: But then there's
some limit with what

1048
00:47:30,820 --> 00:47:34,260
your computer can reconstruct.

1049
00:47:34,260 --> 00:47:36,646
I mean, it's just
infinitely many

1050
00:47:36,646 --> 00:47:40,430
tumbling orientations
or something.

1051
00:47:40,430 --> 00:47:45,750
EDWARD BRIGNOLE: Yeah, it's
a tough experiment to do.

1052
00:47:45,750 --> 00:47:50,514
The other issue is
compressing all your dose

1053
00:47:50,514 --> 00:47:51,930
into a pretty short
amount of time

1054
00:47:51,930 --> 00:47:56,070
so that you basically obliterate
the molecule in this field

1055
00:47:56,070 --> 00:48:01,169
of view but you
capture the image of it

1056
00:48:01,169 --> 00:48:03,460
faster than it's tumbling,
say, or something like that.

1057
00:48:03,460 --> 00:48:04,126
So I don't know.

1058
00:48:04,126 --> 00:48:06,270
Maybe it depends on
its tumbling rate.

1059
00:48:06,270 --> 00:48:07,440
Yeah, something like that.

1060
00:48:07,440 --> 00:48:11,470
But yeah, I think it's
an exciting time for EM

1061
00:48:11,470 --> 00:48:14,600
right now because now
there's new detectors.

1062
00:48:14,600 --> 00:48:17,050
There's actually some
other examples of advances

1063
00:48:17,050 --> 00:48:19,120
that I put on that
slide that allow

1064
00:48:19,120 --> 00:48:22,990
us to look at smaller things,
potentially specimens that

1065
00:48:22,990 --> 00:48:25,330
are still hydrated.

1066
00:48:25,330 --> 00:48:25,830
Yeah.

1067
00:48:28,727 --> 00:48:30,560
I don't know if my email
address is on this,

1068
00:48:30,560 --> 00:48:32,140
but if you come
across that paper

1069
00:48:32,140 --> 00:48:36,170
or see anything like that,
feel free to bounce it to me.

1070
00:48:36,170 --> 00:48:36,670
All right.

1071
00:48:36,670 --> 00:48:38,950
So through the
methods that I just

1072
00:48:38,950 --> 00:48:42,160
described to you, basically from
the fatty acid synthase image

1073
00:48:42,160 --> 00:48:46,180
that we looked at a moment ago,
we can sort out some images.

1074
00:48:46,180 --> 00:48:47,619
And if you look
at this image, it

1075
00:48:47,619 --> 00:48:50,160
looks a lot like that crystal
structure I showed you earlier.

1076
00:48:50,160 --> 00:48:51,660
There's the legs
you can clearly see

1077
00:48:51,660 --> 00:48:53,740
in the average and
the processing enzymes

1078
00:48:53,740 --> 00:48:55,320
in the upper proportion.

1079
00:48:55,320 --> 00:48:57,100
Then we could sort
out a whole bunch

1080
00:48:57,100 --> 00:48:59,730
of other different classes.

1081
00:48:59,730 --> 00:49:01,900
And these puzzled us at first.

1082
00:49:01,900 --> 00:49:05,470
They're kind of fun to look
at because we thought maybe

1083
00:49:05,470 --> 00:49:06,556
it's winking at you.

1084
00:49:06,556 --> 00:49:07,930
It's got one eye
open and one eye

1085
00:49:07,930 --> 00:49:09,650
closed or like the other one.

1086
00:49:09,650 --> 00:49:12,400
And we described these
as different views.

1087
00:49:12,400 --> 00:49:17,330
This looked like it had a
pirate's hat on or something.

1088
00:49:17,330 --> 00:49:20,980
But one of the other things
that puzzled us at first too is,

1089
00:49:20,980 --> 00:49:22,510
this lower portion
of the structure

1090
00:49:22,510 --> 00:49:24,790
here, it looks like
maybe we were getting

1091
00:49:24,790 --> 00:49:28,000
some sort of proteolysis
and these malonyl acetyl

1092
00:49:28,000 --> 00:49:31,000
transferases at the
legs, we thought maybe

1093
00:49:31,000 --> 00:49:33,280
they were getting cut off.

1094
00:49:33,280 --> 00:49:35,860
So we were relieved
when we generated the 3D

1095
00:49:35,860 --> 00:49:38,134
reconstructions then,
that these actually

1096
00:49:38,134 --> 00:49:39,300
weren't getting cleaved off.

1097
00:49:39,300 --> 00:49:42,650
They're just rotated
90 degrees on the grid

1098
00:49:42,650 --> 00:49:44,890
and coming out towards us.

1099
00:49:44,890 --> 00:49:46,600
I'll say a quick
word now about--

1100
00:49:51,900 --> 00:49:53,920
have you read any
crystallography papers yet

1101
00:49:53,920 --> 00:49:55,757
for class when they
talk about resolution

1102
00:49:55,757 --> 00:49:57,340
of the structure,
what kinds of things

1103
00:49:57,340 --> 00:50:01,110
you can see in the structures?

1104
00:50:01,110 --> 00:50:06,040
So in crystallography, you
have a defined resolution limit

1105
00:50:06,040 --> 00:50:09,400
based on the highest angle of
scattering data you collect.

1106
00:50:09,400 --> 00:50:11,677
So it's defined
in the experiment.

1107
00:50:11,677 --> 00:50:12,760
In EM, we don't have that.

1108
00:50:12,760 --> 00:50:15,880
We just have images.

1109
00:50:15,880 --> 00:50:19,780
So the way we calculate
resolution in EM

1110
00:50:19,780 --> 00:50:21,500
is we would take our dataset--

1111
00:50:21,500 --> 00:50:23,920
so the data that went
into this reconstruction

1112
00:50:23,920 --> 00:50:26,410
here if there is 1,000
particles-- we'd split it

1113
00:50:26,410 --> 00:50:29,320
into two subdatasets,
one with randomly

1114
00:50:29,320 --> 00:50:30,820
selected 500 particles
and the other

1115
00:50:30,820 --> 00:50:32,380
with another randomly
selected 500.

1116
00:50:32,380 --> 00:50:34,300
And we'd generate a
reconstruction from both,

1117
00:50:34,300 --> 00:50:35,700
from each of those.

1118
00:50:35,700 --> 00:50:37,450
And we'd compare those,
the reconstruction

1119
00:50:37,450 --> 00:50:39,366
of this half of the data
to the reconstruction

1120
00:50:39,366 --> 00:50:41,412
from this half of the
data and see how similar

1121
00:50:41,412 --> 00:50:42,370
they are to each other.

1122
00:50:42,370 --> 00:50:44,740
And that's how we would
figure out resolution by EM.

1123
00:50:44,740 --> 00:50:47,980
So there's some
problems with that.

1124
00:50:47,980 --> 00:50:53,930
Can anybody think of one
way this would be biased

1125
00:50:53,930 --> 00:50:55,930
or any way that this would
be biased if you just

1126
00:50:55,930 --> 00:50:57,700
take your data, split
it into two halves,

1127
00:50:57,700 --> 00:50:59,500
reconstruct it, compare the two?

1128
00:51:02,050 --> 00:51:09,655
For one, it's sort of like
if you have one person

1129
00:51:09,655 --> 00:51:11,280
do the experiment
and they do it again,

1130
00:51:11,280 --> 00:51:13,100
but nobody else can do it.

1131
00:51:13,100 --> 00:51:14,880
There is a bias in--

1132
00:51:14,880 --> 00:51:17,310
you're taking the
exact same approach

1133
00:51:17,310 --> 00:51:19,150
to initialize both
of these experiments.

1134
00:51:19,150 --> 00:51:22,410
They're both going to converge
to the same local minima.

1135
00:51:22,410 --> 00:51:29,080
So you could be
precisely wrong and have

1136
00:51:29,080 --> 00:51:30,610
a false high resolution.

1137
00:51:30,610 --> 00:51:32,975
So typically, what
you'll see in EM

1138
00:51:32,975 --> 00:51:35,430
papers is a curve like
this, where you basically

1139
00:51:35,430 --> 00:51:38,790
are comparing the two half
reconstructions to each other,

1140
00:51:38,790 --> 00:51:40,410
one from first half
of the dataset,

1141
00:51:40,410 --> 00:51:41,760
the other from the other
half of the dataset.

1142
00:51:41,760 --> 00:51:43,830
You compare them, and
then you look at, say,

1143
00:51:43,830 --> 00:51:45,672
at low resolution,
how similar are they.

1144
00:51:45,672 --> 00:51:47,130
Add a little bit
higher resolution,

1145
00:51:47,130 --> 00:51:48,590
how similar are they?

1146
00:51:48,590 --> 00:51:51,330
If you go to, in this case,
20 angstrom resolution,

1147
00:51:51,330 --> 00:51:52,330
how similar are they?

1148
00:51:52,330 --> 00:51:54,121
And in this case,
they've got a correlation

1149
00:51:54,121 --> 00:51:58,420
of about 10% or 15%.

1150
00:51:58,420 --> 00:52:00,600
So in the case of
this paper, the way

1151
00:52:00,600 --> 00:52:04,217
we reported the
resolution was when

1152
00:52:04,217 --> 00:52:06,300
the correlation between
the two halves of the data

1153
00:52:06,300 --> 00:52:07,740
fell to about 50%.

1154
00:52:07,740 --> 00:52:10,290
So we reported a resolution
of about 30 angstroms

1155
00:52:10,290 --> 00:52:15,090
for these structures,
but like I said,

1156
00:52:15,090 --> 00:52:17,010
that doesn't necessarily
mean they're right.

1157
00:52:17,010 --> 00:52:19,506
And in our case, the
advantage we have

1158
00:52:19,506 --> 00:52:21,130
was that there was
a crystal structure.

1159
00:52:21,130 --> 00:52:23,171
So if we just dropped the
crystal structure right

1160
00:52:23,171 --> 00:52:26,492
into the EM reconstruction, that
looks like a pretty good match.

1161
00:52:26,492 --> 00:52:28,450
And one thing that we
didn't do for this paper,

1162
00:52:28,450 --> 00:52:33,500
but people sometimes
do is, instead

1163
00:52:33,500 --> 00:52:36,000
of comparing half of our data
to the other half of our data,

1164
00:52:36,000 --> 00:52:38,550
we could have compared our data
versus the crystal structure

1165
00:52:38,550 --> 00:52:42,390
and come up with a
similar curve to compare

1166
00:52:42,390 --> 00:52:43,950
our data to this
high resolution data

1167
00:52:43,950 --> 00:52:47,470
and see where things fall off.

1168
00:52:47,470 --> 00:52:49,660
So in these
reconstructions, there's

1169
00:52:49,660 --> 00:52:51,970
lots of different conformations.

1170
00:52:51,970 --> 00:52:54,850
You can see the lower portion
swinging back and forth,

1171
00:52:54,850 --> 00:52:59,820
the upper portion twisting
relative to the lower portion.

1172
00:52:59,820 --> 00:53:02,300
And then, you see this
other conformational change.

1173
00:53:02,300 --> 00:53:03,970
So we take the
arms off of the end

1174
00:53:03,970 --> 00:53:06,010
and look at what's happening
in the middle here.

1175
00:53:06,010 --> 00:53:07,780
The enoyl reductase
and dehydratase

1176
00:53:07,780 --> 00:53:10,979
are rotating like this
relative to each other.

1177
00:53:10,979 --> 00:53:13,020
So one side opens up while
the other side closes.

1178
00:53:13,020 --> 00:53:15,900
They sort of cross over
each other like that.

1179
00:53:15,900 --> 00:53:18,580
And so when one side
rotates, they sort of rotate,

1180
00:53:18,580 --> 00:53:21,130
but then one side tightens
up while the other side

1181
00:53:21,130 --> 00:53:22,570
comes loose.

1182
00:53:22,570 --> 00:53:28,000
So we wanted to know how
these related to catalysis.

1183
00:53:28,000 --> 00:53:33,370
So I guess if you've
looked at the paper--

1184
00:53:33,370 --> 00:53:36,100
I'm going to try to speed
up here a little bit--

1185
00:53:36,100 --> 00:53:38,442
what we did was we
looked at some mutants

1186
00:53:38,442 --> 00:53:40,150
in the presence and
absence of substrates

1187
00:53:40,150 --> 00:53:41,786
to look at how this--

1188
00:53:41,786 --> 00:53:43,660
we've got all these
different conformations--

1189
00:53:43,660 --> 00:53:47,500
how the frequency that we
see different conformations

1190
00:53:47,500 --> 00:53:48,970
changes.

1191
00:53:48,970 --> 00:53:51,670
And I'll sort of
cut to the chase.

1192
00:53:51,670 --> 00:53:53,482
I think in the paper,
there is histograms,

1193
00:53:53,482 --> 00:53:55,690
but I think the pie charts
are a little more telling.

1194
00:53:55,690 --> 00:53:58,480
But basically, what happens
is if you add substrates,

1195
00:53:58,480 --> 00:54:00,730
the conformation that
becomes most prevalent

1196
00:54:00,730 --> 00:54:03,800
is the one that's
represented in blue here.

1197
00:54:03,800 --> 00:54:06,184
So if I go back, you've
got these, basically,

1198
00:54:06,184 --> 00:54:08,350
four different categories
where the lower portion is

1199
00:54:08,350 --> 00:54:11,140
perpendicular or
parallel and then

1200
00:54:11,140 --> 00:54:13,630
whether the upper portion has
this asymmetric appearance

1201
00:54:13,630 --> 00:54:14,230
or not.

1202
00:54:14,230 --> 00:54:17,140
And the one thing that
jumps out right away

1203
00:54:17,140 --> 00:54:18,820
is that add
substrates and you get

1204
00:54:18,820 --> 00:54:21,430
lots of asymmetry
in the upper portion

1205
00:54:21,430 --> 00:54:22,930
and the lower
portion in parallel

1206
00:54:22,930 --> 00:54:25,650
with the bottom portion.

1207
00:54:25,650 --> 00:54:26,320
All right.

1208
00:54:26,320 --> 00:54:29,150
So why would that be?

1209
00:54:29,150 --> 00:54:31,007
So if we look at one
of these conformations

1210
00:54:31,007 --> 00:54:33,340
where the lower portion's
parallel to the upper portion,

1211
00:54:33,340 --> 00:54:37,180
the upper portion has this
asymmetric appearance.

1212
00:54:37,180 --> 00:54:39,730
What might this reaction
chamber be good at doing?

1213
00:54:49,873 --> 00:54:56,040
So these enzymes in the lower
portion, the ketoacyl synthase

1214
00:54:56,040 --> 00:54:57,570
and the
malonyltransferase, they've

1215
00:54:57,570 --> 00:55:01,830
come up close to where the
acyl carrier protein would be.

1216
00:55:01,830 --> 00:55:04,470
And at the same time,
this side is closed off,

1217
00:55:04,470 --> 00:55:07,710
so it would have a harder time
doing processing on this side.

1218
00:55:07,710 --> 00:55:11,442
So at the same time over
here, this side's opened up.

1219
00:55:11,442 --> 00:55:12,900
The acyl carrier
protein can easily

1220
00:55:12,900 --> 00:55:14,274
get in here to do
the processing,

1221
00:55:14,274 --> 00:55:16,290
but these enzymes
over here are out

1222
00:55:16,290 --> 00:55:19,230
of reach of the
elongation enzymes.

1223
00:55:19,230 --> 00:55:20,730
So what this means
is one side could

1224
00:55:20,730 --> 00:55:24,130
be elongating while the
other side's processing.

1225
00:55:24,130 --> 00:55:26,240
And then the structures
are symmetric.

1226
00:55:26,240 --> 00:55:27,992
So if you flip it
around, then this side,

1227
00:55:27,992 --> 00:55:29,700
once it's done
elongating, could process.

1228
00:55:29,700 --> 00:55:31,116
And then this side
could elongate.

1229
00:55:31,116 --> 00:55:34,221
So it's kind of cool because
it can sort of balance out

1230
00:55:34,221 --> 00:55:35,970
what it's doing from
one side to the next.

1231
00:55:35,970 --> 00:55:38,136
And then we have these
confirmations where the lower

1232
00:55:38,136 --> 00:55:39,210
portion is perpendicular.

1233
00:55:39,210 --> 00:55:40,710
And remember, at
the beginning I had

1234
00:55:40,710 --> 00:55:42,430
said that we know
that this acyl carrier

1235
00:55:42,430 --> 00:55:44,310
protein can elongate
with the enzymes

1236
00:55:44,310 --> 00:55:47,010
in the opposite portion.

1237
00:55:47,010 --> 00:55:50,160
So because of the
symmetry in the system

1238
00:55:50,160 --> 00:55:52,230
and also the resolution
of the structures,

1239
00:55:52,230 --> 00:55:54,480
we can't tell the difference
between whether the lower

1240
00:55:54,480 --> 00:55:57,600
portion is flipped 180
degrees relative to the top

1241
00:55:57,600 --> 00:56:00,690
or not because it
would look the same.

1242
00:56:00,690 --> 00:56:04,650
But the fact that we can
see these go 90 degrees

1243
00:56:04,650 --> 00:56:08,880
is suggestive that it could
probably unravel and go

1244
00:56:08,880 --> 00:56:10,030
the rest of the way around.

1245
00:56:10,030 --> 00:56:12,030
So in the crystal structure,
the way it had them

1246
00:56:12,030 --> 00:56:15,700
is, they were coiled like this.

1247
00:56:15,700 --> 00:56:17,400
And so it's pretty
easy to imagine

1248
00:56:17,400 --> 00:56:19,900
that they would just uncoil.

1249
00:56:19,900 --> 00:56:21,720
One other line of
evidence that I think

1250
00:56:21,720 --> 00:56:24,360
is telling-- so our
collaborators made

1251
00:56:24,360 --> 00:56:27,797
a mutant that has all
of the active sites

1252
00:56:27,797 --> 00:56:30,130
and the acyl carrier protein
knocked out of one subunit.

1253
00:56:33,230 --> 00:56:36,510
So there is one subunit totally
wild type and the other subunit

1254
00:56:36,510 --> 00:56:40,070
that's totally dead.

1255
00:56:40,070 --> 00:56:41,820
So the interesting
thing about this mutant

1256
00:56:41,820 --> 00:56:51,080
is it has to do the condensation
reaction, the elongation,

1257
00:56:51,080 --> 00:56:54,915
in this conformation,
sort of crossed over.

1258
00:56:54,915 --> 00:56:57,510
But to pick up its starter
unit or elongating unit,

1259
00:56:57,510 --> 00:57:00,510
it has to coil back
around for this.

1260
00:57:00,510 --> 00:57:04,680
And we know based on
the rate of this enzyme,

1261
00:57:04,680 --> 00:57:10,370
that this probably happens
about 100 times per minute.

1262
00:57:10,370 --> 00:57:14,740
There's a functional catalytic
event that happens 100 times

1263
00:57:14,740 --> 00:57:15,240
a minute.

1264
00:57:15,240 --> 00:57:18,090
So it probably is sampling
these much more rapidly.

1265
00:57:18,090 --> 00:57:18,795
Sure.

1266
00:57:18,795 --> 00:57:21,345
AUDIENCE: So this
isn't compensating

1267
00:57:21,345 --> 00:57:25,197
for when you knock out one half
that's naturally [INAUDIBLE]

1268
00:57:25,197 --> 00:57:27,030
EDWARD BRIGNOLE: Yeah,
that's what we think.

1269
00:57:27,030 --> 00:57:28,650
It's sort of naturally
sampling both sides.

1270
00:57:28,650 --> 00:57:30,691
It's interesting to think
about because let's say

1271
00:57:30,691 --> 00:57:35,919
this side picks up acetate and--

1272
00:57:35,919 --> 00:57:37,710
well, let me think
about this for a second.

1273
00:57:37,710 --> 00:57:41,250
But if one side is
ready to elongate

1274
00:57:41,250 --> 00:57:44,800
and the other one's
got a starter unit,

1275
00:57:44,800 --> 00:57:48,780
and this side over here
is already loaded with--

1276
00:57:48,780 --> 00:57:51,360
so basically, this will
pick up an acetyl group

1277
00:57:51,360 --> 00:57:53,457
and transfer it to
the ketoacyl synthase.

1278
00:57:53,457 --> 00:57:55,290
And then it comes back,
and then it picks up

1279
00:57:55,290 --> 00:57:56,840
an acetylic group again.

1280
00:57:56,840 --> 00:57:59,240
So then it would
be stuck because it

1281
00:57:59,240 --> 00:58:02,660
would be trying to extend
an acetyl with an acetyl.

1282
00:58:02,660 --> 00:58:04,242
But it needs a malonyl.

1283
00:58:04,242 --> 00:58:05,700
So what it could
do is flip around,

1284
00:58:05,700 --> 00:58:08,630
sample the other side, which
might have a malonyl group,

1285
00:58:08,630 --> 00:58:12,000
to continue on on that side
and then come back around.

1286
00:58:12,000 --> 00:58:14,190
And it could transfer
the acetyl group on.

1287
00:58:14,190 --> 00:58:17,160
So it'd allow maybe
one way that it

1288
00:58:17,160 --> 00:58:20,520
doesn't have to
necessarily go backwards,

1289
00:58:20,520 --> 00:58:22,410
though the malonyl
acetyl transferase can

1290
00:58:22,410 --> 00:58:23,280
function in reverse.

1291
00:58:23,280 --> 00:58:27,296
So that's another thing, is it
won't get stuck if everything's

1292
00:58:27,296 --> 00:58:29,670
all loaded up with acetate
because the malonyltransferase

1293
00:58:29,670 --> 00:58:32,790
will run backwards to
cut the acetates off.

1294
00:58:32,790 --> 00:58:35,800
But this would be
one way to maybe not.

1295
00:58:35,800 --> 00:58:37,440
It wouldn't have
to necessarily rely

1296
00:58:37,440 --> 00:58:38,910
on going backwards
to get unstuck.

1297
00:58:38,910 --> 00:58:41,051
It could just twist around.

1298
00:58:41,051 --> 00:58:41,550
All right.

1299
00:58:41,550 --> 00:58:45,460
So I think we'll just finish up
with a quick movie that shows

1300
00:58:45,460 --> 00:58:46,710
these different conformations.

1301
00:58:46,710 --> 00:58:49,610
So the bottom parts
picks up a substrate,

1302
00:58:49,610 --> 00:58:51,840
elongates it, goes up
here to do the processing.

1303
00:58:51,840 --> 00:58:54,120
Meanwhile down here,
it's elongating.

1304
00:58:54,120 --> 00:58:59,100
You can see how up here in the
upper portion, the separation

1305
00:58:59,100 --> 00:59:02,216
of where the dehydratase
and enoyl reductase is

1306
00:59:02,216 --> 00:59:03,840
so the acyl carrier
protein can fit in.

1307
00:59:11,536 --> 00:59:13,520
Here we go.

1308
00:59:13,520 --> 00:59:16,350
So then there was that bonus
question at the beginning.

1309
00:59:16,350 --> 00:59:18,390
I know you guys
probably have to run.

1310
00:59:18,390 --> 00:59:20,360
So the bonus question
at the beginning is,

1311
00:59:20,360 --> 00:59:22,943
what is that structural domain
that's-- the methyltransferase,

1312
00:59:22,943 --> 00:59:24,811
where did that come from.

1313
00:59:24,811 --> 00:59:25,310
Any ideas?

1314
00:59:28,270 --> 00:59:30,880
You guys have talked a little
bit about polyketide synthases

1315
00:59:30,880 --> 00:59:32,760
yet?

1316
00:59:32,760 --> 00:59:34,820
A lot of them have
domain architectures

1317
00:59:34,820 --> 00:59:37,480
identical to our
fatty acid synthase

1318
00:59:37,480 --> 00:59:40,010
with functional
methyltransferases.

1319
00:59:40,010 --> 00:59:43,490
So it seems like we probably
picked up our fatty acid

1320
00:59:43,490 --> 00:59:45,950
synthase from something
like a lovastatin synthase

1321
00:59:45,950 --> 00:59:49,350
or something like that.

1322
00:59:49,350 --> 00:59:51,830
So it's interesting
to think about.

1323
00:59:51,830 --> 00:59:56,585
And then we didn't need it,
so it's now not functional.

1324
00:59:56,585 --> 00:59:57,830
All right.

1325
00:59:57,830 --> 00:59:59,030
Cool.

1326
00:59:59,030 --> 01:00:00,880
Thanks guys.