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  • Hi, my name is Eva Nogales, I'm a professor of molecular and cell biology at UC-Berkeley,

  • a Howard Hughes Medical Institute Investigator and a senior faculty scientist at the Lawrence Berkeley national lab.

  • And today, I would like to give you an introduction to what is my favorite visualization technique

  • to see cellular and molecular details in biology.

  • This is electron microscopy. In my lab we use this method to visualize processes

  • of cellular self-assembly and molecular machines that are involved in nucleic acid transactions.

  • And I will be using some of these as examples. I'm going to start by giving you

  • an introduction to the physics behind how electrons interact with matter,

  • how the electron microscope is able to generate

  • contrast in visualizing sample

  • and then give you examples of biological molecules and cells

  • and how the images are generated, how they look and

  • how we go into farther processing to learn more

  • about structure and function in cell biology.

  • So, the first thing that I would like to tell you is that electron microscopy comes in two very different flavors.

  • SEM, or scanning electron microscopy, is a method that uses

  • focused beams of low-energy electrons to raster along a bulky object

  • and give you a low resolution rendition of the surface

  • of the object. So this is an example of such an image.

  • It's a crab larvae and it looks very pretty because it has added false color,

  • but there's no color whatsoever in electron microscopy, so that's the first thing that you

  • have to remember. This is more like the real image

  • Let me give you another one, this is of dust mites,

  • these are nasty things that cause allergies certainly they do only.

  • And I want you to pay attention to the scale bar here,

  • which is two hundred microns, this is about the size of these organisms.

  • You could certainly crank up the magnification in this type of microscopy

  • and get to visualize individual cells.

  • So this is an image now of a mouse oviduct.

  • And these kind of tissue has lots of cells that have these cilia that they use to create fluid motion

  • and allow the sperm and the egg to encounter one another.

  • Notice that the scale bar now is 2 microns, so we're really seeing cellular details right now.

  • I would like to concentrate from now on on the other electron microscopy technique,

  • which is Transmission Electron Microscopy, or TEM.

  • This uses very different principles and what it gives you is a projection

  • images at much higher resolution of thin samples that are introduced into the microscope and there's possibility here

  • to get even to an atomic resolution,

  • as I will tell you in a second.

  • So this particular example comes from a section that was taken from the flagella of a sperm,

  • the one that could have been coming up the oviduct that I showed you before.

  • So, in this section, what we see is the axoneme,

  • a structure that goes all the way along the flagella,

  • it's made up by microtubules, which are organized into this beautiful structure.

  • Now, microtubules are made by self-assembly of the protein tubulin.

  • and you can actually purify tubulin, typically from mammalian brain, like cow brain,

  • and reconstitute the process of microtubule assembly in vitro, in the test tube.

  • And this is now a TEM image of such individual microtubules.

  • And this image now contains information at the atomic level.

  • The scale bar in the image that I showed you before

  • was not microns, it was nanometers.

  • And what you see here, the thickness of one of these microtubules is 250 angstroms, or 25 nanometers.

  • In fact, this methodology, has been used to obtain information about the structure of the tubulin protein itself.

  • And what I show you here is a density map in blue inside the protein molecule, where these yellow lines represent the polypeptide chain

  • going along it. And every one of these squares in blue corresponds now to one angstrom.

  • So the resolution here is much higher, is at the atomic level.

  • So, I want to give you more examples from microtubules, which are one of my favorite biological samples in a minute.

  • But before that, I want to go back to very basic principles of how electrons interact with matter.

  • In the transmission electron microscope, electrons have very high energies,

  • of the order of hundreds of kiloelectron volts.

  • That means that they are moving at close to relativistic speed,

  • They are moving so fast that sometimes they basically go through matter without even noticing.

  • So, in here, what you can see are electrons going past an atom and some of them just go through and simply don't interact.

  • They don't see anything. Some of them, and these are very important for generating the image,

  • are going to go through and bounce off the nucleus of the atom and be bent.

  • This is a process of elastic scattering, in which electrons don't change energy, don't change speed,

  • but change direction. And this is what we call the good scattering

  • and is going to be used to generate contrast in the image, in the TEM.

  • Unfortunately, there's another process of scattering,

  • the bad scattering, and these are electrons in your primary beam generated in the microscope

  • that go through the atom and interact with the electrons in your sample

  • and in doing so, they lose energy of their own and they pass it to your sample.

  • These inelastically scattered electrons

  • are going to only contribute to the noise in your image,

  • while at the same time, really damaging your sample.

  • Ok, so, the TEM is going to be able to visualize your sample

  • by using these elastically scattered electrons to generate contrast in the image.

  • And the way this is done is in two different manners.

  • The first type of contrast is called amplitude contrast and is the one that is used to visualize sections of cells.

  • Like this one, this is a section through the root of a maize plant

  • and you can see the variety of internal organelles in here.

  • Amplitude contrast works very much like an X-ray

  • that you get at the doctor's, where more X-rays are being absorbed by your bones

  • than by the rest of your soft tissue,

  • so the bones appear darker in the image.

  • Actually, what I'm showing you here is the positive of the image.

  • What the doctor shows you is the negative, which looks like this.

  • Ok, so in the microscope, in the TEM, electrons can be either absorbed by the sample or

  • otherwise they can be elastically scattered

  • and then we can remove the elastically scattered electrons by means of an aperture.

  • The aperture is placed after the objective lens

  • and what is does is it allows the unscattered electrons to go through, but it blocks the elastically scattered ones.

  • And that generates contrast in the image,

  • basically making regions where lots of scattering happens, because there's more density, appear dark

  • and regions where there was less scattering appear bright.

  • This type of contrast is great to visualize sections of cells,

  • but it falls really short when you're trying to get high resolution information

  • on proteins and other macromolecular complexes.

  • So, for that, we need phase contrast. This is the contrast

  • that is generated in these types of images, now, of purified protein and protein complexes.

  • In this case, the objective aperture of the microscope is utilized to make the scattered, elastically scattered electrons,

  • and the unscattered electrons interfere,

  • generating contrast in that manner.

  • This is based on the principle that relativistic electrons basically can be seen as waves

  • and that when they go through matter, the wave front will suffer a phase shift

  • and this is what will ultimately cause the interference, which will mix with the unscattered wave front.

  • I'm not going to into any more physics, rather, I will now show you how an electron microscope looks like.

  • Ok, this is what I would call a middle of the range electron microscope.

  • The whole story starts up here with the electron gun,

  • which is where the electrons are produced.

  • They shoot down the column, which is maintained at very high vacuum

  • so as to minimize the scattering of electrons by residual air.

  • At each stage you have electromagnetic lenses that are used to deflect the path of the electrons

  • Earlier on, there's two condenser lenses and these

  • control the illumination of the sample, how bright and how large is the area that's being illuminated

  • and then comes the most important lens in the scope,

  • which is right in the middle. It's the objective lens, this is the one that

  • will combine the scattered and non-scattered electrons to give you contrast in the image.

  • And the other thing that happens right here

  • is that's where the sample goes in.

  • In this case, this is a cryo sample that is being maintained at liquid nitrogen temperature

  • by being in contact with this dewer, with liquid nitrogen.

  • So, after the objective lens come a number of intermediate lenses

  • that are utilized to change the magnification from 50 times to something as high as 400,000 times.

  • The image can then be observed in a phospo screen, directly on a TV

  • and ultimately recorded for further data processing,

  • either on photographic film or on a CCD camera.

  • The one thing that I would like to show you is an electron microscope grid.

  • It's right there on my finger. This metal grid is coated by a thin layer of carbon

  • and then the sample goes in there.

  • And we need extremely little amount of sample.

  • We use, say for purified protein complexes, concentrations that are on the nanomolar,

  • sub-micromolar range and we use a very small drop, the size of small tear,

  • that goes right in there. And then gets blotted to a thin layer

  • before it's very quickly frozen.

  • The grid, all of this is done, under liquid nitrogen or nitrogen gas and then in the holder

  • and then it is introduced in the electron microscope.

  • But with very, very little sample, we can still get millions of occurrence of the complex

  • we are interested in and take many, many images and hopefully get the structure we need.

  • I get often asked what kind of resolution can the TEM reach.

  • And in fact, microscopes like the one that I showed you is capable of obtaining images

  • with atomic detail. This is such an example,

  • this corresponds to a thin, nanocrystal of silica,

  • where each one of these dots are actually atoms that have been visualized in this image.

  • In fact, a state of the art electron microscope can reach resolutions

  • beyond a single angstrom. So, very high resolutions are achievable,

  • as long as the sample is not radiation sensitive.

  • So, this sample was able to withstand thousands of electrons going through it in each square angstrom.

  • in the sample, without damage. Unfortunately, that is not the case for biological material,

  • which is extremely sensitive to radiation.

  • It was only a few years ago that people thought that high-resolution information

  • from biological materials would never be reached

  • because the sample will vaporize before images were finally collected.

  • If that had been the case, I would be very sad, would not have a job

  • and I would be not talking to you today.

  • But, fortunately, there is more than one way in which we can trick nature

  • and obtain high resolution information from our biological samples.

  • So, let me review with you what the problems are that are unique to biological, organic samples.

  • First of all, all biological material lives in aqueous solution

  • and by definition they hate the high vacuum in the column of the electron microscope.

  • The other thing is that the atoms that biological materials are made of, nitrogen, carbon, oxygen,

  • have basically the same scattering power as the water that is surrounding them.

  • So, they basically have very low intrinsic contrast.

  • And ultimately, and most importantly, they are very radiation sensitive.

  • When inelastic scattering occurs, the sample gets ionized,

  • generating radicals, that then move around the sample

  • and break all the bonds and basically make the whole thing explode.

  • Ok, so, how do we overcome these problems?

  • There's two solutions. The first one to occur historically was negative staining.

  • In this case, your sample is embedded in a low concentration of a salt solution

  • of a very heavy atom, typically uranium.

  • The sample is embedded in this solution.

  • The solution is then dried to a thin layer

  • and introduced into the electron microscope.

  • Because now there's no water, there's no problem with the vacuum.

  • The heavy atom, the uranium, generates very high contrast

  • so we can overcome the second problem

  • and because now what you're imaging is this cast generated

  • by the stain, rather than the protein

  • you also reduce the problem of radiation damage.

  • The protein may vaporize, but as long as the cast still reproduces its shape,

  • we are ok. So, the big pluses of this methodology are the high contrast in the image,

  • and the fact that it's fast and easy.

  • So, Berkeley undergraduate students can come to my lab and in a few weeks, they're ready and taking beautiful images

  • using this methodology. Now, there are minuses,

  • of course. The minus, the big minus, is that artifacts are readily possible.

  • This is due to the fact that in some cases, the stain cannot penetrate inside the protein

  • or cases where, as you dry the stain, the protein structure may collapse.

  • Even if you have very good preservation, in some cases, the resolution is always limited.

  • It's limited because as you dry the stain, it forms little grains,

  • and that's the ultimate size of anything that you're going to be able to see,

  • which is typically about 15 angstroms.

  • So, the solution, the second optimized solution is to look at unstained, frozen, hydrated samples.

  • This is what we call cryo-electron microscopy. In this case,

  • the sample is embedded in active solution where it is happy,

  • but then is very quickly frozen. It's frozen so fast that the water molecules

  • don't have time to reorganize into a crystal

  • into ice, and therefore remains amorphous. We call that vitrified water.

  • To achieve vitrification, the samples have to be frozen very fast,

  • typically a million degrees per second

  • and then kept at very low temperatures, the temperature of liquid nitrogen.

  • If you do that, this sample can go into the electron microscope without evaporation of the water.

  • So, we avoid the problem of vacuum altogether.

  • So, the sample is hydrated, but in a solid state that can withstand the high vacuum.

  • Now, because there's no stain, these samples do suffer from low contrast,

  • and we're going to have to overcome that by other means,

  • I'll tell you about that later.

  • Radiation sensitivity is limited because now the very low temperatures means that radicals that were generated

  • through the process of inelastic scattering are not able to move very fast.

  • So, that radiation damage is minimized.

  • Not eliminated, but reduced.

  • So, the pluses of this technique is that the preservation is extremely good

  • because basically you have preserved even the aqueous layer that surrounds your protein.

  • Because there's no stain and there's no graininess, high resolution is in principle achievable.

  • In fact, in some cases, if you have the right experimental set-up,

  • you can even obtain time resolution.

  • You can time a certain biological process to be triggered during the process of vitrification and trap intermediates.

  • Now, minuses. This is a much more technically demanding technique,

  • so, undergraduates in my lab rarely get to a point where they are feeling very comfortable about doing cryo-EM.

  • It takes many months, if not years, to really master.

  • The other problem is the contrast, as I told you.

  • There are ways of enhancing the contrast in the electron microscope,

  • but they always tend to come at a price.

  • So, we mostly we deal with that computationally.

  • The other problem is that although we have minimized radiation,

  • still the sample remains sensitive. So, we have to use

  • low doses, typically 10-20 electrons per angstrom squared.

  • And that means that the images are going to be very noisy.

  • So, let me give you an example how the same sample. the same biological material

  • looks like in negative stain versus cryo-EM.

  • So, what you see here on the top is an image of a microtubule that is surrounded by rings

  • that are made of a kinetochore protein.

  • The kinetochores are the structures by which microtubules interact with chromosomes

  • in a process called mitosis, by which genetic material is separated.

  • So, here is that sample in negative stain.

  • This is uranyl acetate that is used to generate this very high contrast.

  • Notice that the proteins appear white, while the stain around it appears black.

  • The image below is exactly the same sample, but now what you're looking at is just the contrast

  • of the protein on a background of water.

  • And the image appears a lot cleaner

  • because in here we can see every individual protein, even those here in the background

  • that have not self-assembled into these beautiful structures,

  • while this in here is basically invisible.

  • On the other hand, what we really present very beautifully in the cryo-EM image is the cylindrical shape of the microtubule

  • and the circular shape of the ring,

  • which allows us to obtain ultimately the structure in high detail and with high reliability.

  • Ok, so, let me now go back to the electron microscope

  • to show you what is a true state of the art TEM machine.

  • Alright, now, this beast is what I would call a state of the art transmission electron microscope.

  • You can see that the column is both longer and wider.

  • This is because the electrons that are being emitted by the electron gun

  • have higher energy; as they're moving faster, they need bigger electromagnetic lenses to deflect them.

  • This microscope has two special, very unique things.

  • One is the sample goes here, this is what we call the stage,

  • and this sample is being maintained at liquid helium temperature.

  • That's very much close to absolute zero, minus 270 degrees centigrade.

  • So, that reduces radiation damage

  • and also, the whole mechanical stage makes these samples very, very stable.

  • And it makes a difference if the sample really doesn't move when you are taking the picture.

  • Now, the other thing that is very important in this microscope and the reason why it is so tall

  • that I have to stand on a ladder is that it has an extra piece right here.

  • This is an in-column energy filter.

  • It works very much like a prism, but for electrons.

  • It spreads them out in a rainbow depending on their energy

  • and that allows us to filter out the inelastically scattered electrons

  • that are contributing only to the noise in the image.

  • This is particularly important when the samples that you're looking at

  • are thick sections of thick cells, where the signal is going to be very low

  • and the amount of inelastically scattered electrons is very large.

  • This will allow us to clean up the image and be able to visualize things that otherwise would be invisible.

  • Ok, this is a good time now to recap

  • and think of the basic principles of how to generate images of biological materials.

  • In most cases, we start with a purified sample of your biological material of interest.

  • This one is the deposited on a substrate in the EM grid that I showed you before,

  • typically covered with carbon, and it's either embedded in negative stain

  • or in a thin layer of vitrified water.

  • Then we pass electrons through it.

  • Some electrons go right through

  • and others are elastically scattered and it will be the interaction of the unscattered and scattered electrons that will give you an image

  • in the electron microscope. But remember,

  • although we start with a 3-dimensional object,

  • what the image in the TEM gives you is a 2-dimensional projection of the object.

  • Remember, this is not a surface like in SEM,

  • it is a projection of the whole structure, but now compacted into two dimensions.

  • Things are really worse than that because the radiation sensitivity of the sample

  • means that we put in very few electrons to generate the image and the image is really noisy.

  • So, this is the true data that we have to deal with.

  • From here, from this noisy, 2-dimensional images, we need to get back to 3-dimensional object

  • in great detail. So, how do we do it?

  • That is, the details are going to depend on the type of sample,

  • but typically involve a process by which many images of the object in the same orientation

  • are identified, aligned and averaged to recover the signal so that now we have things that look more like that.

  • If we can get these type of images now,

  • but of the object in different orientations,

  • then they can be combined as long as we find out the relative orientation between them

  • to move from 2D to 3D and recover a structure.

  • This process is what we call reconstruction

  • and how each one of these two steps are carried out depends

  • very much on what type of sample do you have.

  • So, one type of sample that is ideal, but comes very rarely,

  • is that of 2 dimensional crystals of proteins.

  • In this case, the protein is arranged in a single plan in an ordered lattice

  • that can extend for several microns.

  • with a thickness that is just a single protein.

  • This kind of sample always falls in the same orientation in the grid,

  • so you know that to get 3-dimensional information it is absolutely required

  • that you do what we call tilting.

  • This means tilting the sample with respect to the electron beam

  • so that we can generate different views of the object.

  • This tilting process is actually experimentally very complex and difficult,

  • but once the data is collected, the computational processing is very simple.

  • And it actually allows you to get to very high resolution fairly fast.

  • This is because the image of these ordered arrays

  • contains very high resolution information, as can be seen in this electron diffraction pattern,

  • from such two dimensional protein crystals which extend to about 3 angstrom resolution.

  • Another type of sample that is really very helpful and great for doing EM are helical arrangements.

  • These can be naturally occurring or they can be artificially produced.

  • Because in a helix, the molecule is in different orientations

  • as you move through the helix, you get different views that are related by the geometry of the helix.

  • So, no tilting is needed and you can actually obtain a full, 3 dimensional reconstruction from a single image

  • although initially, you may have low resolution.

  • Now, these type of methodology is able to get between medium to high resolution,

  • meaning between 10 and 3 angstrom resolution.

  • And, like for crystals, the order in these structures, means that in Fourier space,

  • if you want, in the diffraction pattern, we have reflections that are well-separated

  • and we're filtering, I'm not going to go into details,

  • but the filtering is equivalent of an averaging process.

  • So, the 2D classification and alignment and the 3D reconstruction are very trivial computationally

  • for both of these two samples.

  • However, the most general type of biological sample is not going to be a 2 dimensional crystal

  • and is not going to be organized into a helix.

  • In that case, the type of reconstruction that we do is called single particles.

  • Typically, these objects are going to be randomly oriented in your EM grid

  • and no tilt will generally be needed.

  • The type of resolution that you get is going to depend on the type of sample.

  • For objects that don't have any internal symmetry and that may have floppy regions,

  • the resolution may be very low, on the order of a few nanometers,

  • while for objects with internal symmetry, like is the case for viruses,

  • the resolution can be very high, all the way to 3 or 4 angstroms.

  • In these cases, where there's no supra-molecular arrangement,

  • the computation is really heavy, it takes a big toll of the data processing.

  • So, let me show you some examples, let's go back to microtubules.

  • Microtubules are an example of cytoskeletal self-assembly into helical structures.

  • As I told you, microtubules are made of alpha-beta tubulin,

  • which are represented here by these light and dark cubes.

  • They associate longitudinally, making what we call protofilaments

  • and these associate in parallel, making the wall of the microtubule.

  • From images like these, of this structure, using helical reconstruction procedures,

  • it is possible to obtain structures like this, where each one of these correspond to a tubulin molecule,

  • and you can see details on the secondary structure,

  • the architecture of the molecule, one at a time.

  • It so happened that in the case of tubulin, you can trick it to self-assemble

  • into something different where protofilaments still form, but they associate in an anti-parallel fashion

  • where the structure doesn't close into a tube

  • but rather it grows into what can be considered two dimensional crystals.

  • These are the ones that produced these beautiful diffraction patterns that I showed you,

  • due to the high order in this polymer.

  • And from here, it is possible to obtain atomic resolution information

  • and that's where ribbon diagrams like this that now describe the path of the tubulin chains

  • could be obtained. I just want you notice that this was obtained in the presence of Taxol,

  • which is here shown in yellow. This is an anti-cancer drug that is used to bind to tubulin

  • and stabilize microtubules, make microtubules very stable.

  • And that has stopped the process of cell division

  • and it stops in particular cells that are dividing very fast,

  • those being cancer cells.

  • Typically, microtubules are very highly dynamic

  • and microtubules have been the object of cryo-EM study to describe actually

  • how the process of assembly and disassembly take place.

  • So, what I'm going to show you now is a short animation

  • that describes in very graphic way

  • how we think microtubules undergo the process of assembly and disassembly

  • based on cryo-EM structure of the intermediates that are generated in the process.

  • So, this is a microtubule that has reached a critical state

  • where it's going to lose its stability and is going to start depolymerizing.

  • This is the tubulin structure that I showed you before

  • so that you have an idea of how it arranges into the microtubule.

  • Microtubules break down actually by peeling back and curling of individual protofilaments.

  • The peels are normally very short lived,

  • they break apart and they depolymerize into individual subunits.

  • But we were able to trap them biochemically

  • in order to obtain this structure of tubulin in that conformation.

  • And what we found was that tubulin subunits are normally interacting with a kink,

  • but they are kinked internally and that is what makes it impossible for them to remain stably in the microtubule.

  • As a molecule of GDP is exchanged for a molecule of GTP

  • that re-energizes the tubulin molecule, straightens it out

  • and allows it to now form both longitudinal and lateral contacts

  • in what is, we believe, are the structural intermediates in the process of assembly

  • that is open and outwardly curved.

  • We could again stabilize that polymer by means of low temperatures and a non-hydrolyzable GTP analogue.

  • And this is the structure of what we saw,

  • was that the protofilaments here are paired up and within one pair

  • the interaction is just like protofilaments in the microtubules but between pairs, these interactions have rotated

  • and as this thing grows, it eventually starts rotating around that special interface

  • so that is closes into a tube in a process that can be very highly cooperative,

  • by zipping up of the tube as the protofilaments straighten.

  • So, typically, you would have a microtubule that is growing by addition of tubulin subunits

  • into an open sheet that then closes into a tube.

  • And eventually, this microtubule will grow, will reach a critical step and then will start depolymerizing.

  • And assembly and disassembly will constantly occur, as in the cell.

  • Ok, so I showed you samples, using tubulin, of how helical reconstruction or 2-dimensional crystals

  • are used to obtain high resolution information on a sample.

  • But, in many cases, we have to rely on single particle techniques

  • because these highly ordered structures are not available.

  • So, let me very quickly go through the processing that will have to take place

  • in a single particle project in order to get to the final structure.

  • To start, remember that we have our sample embedded, again, purified sample,

  • molecules, embedded either in stain or vitreous water,

  • that the EM image gives you a two-dimensional projection that is actually very noisy

  • because of the low doses that we can use.

  • Now, from here, what we will do is we'll visualize each one of these occurrence of our, say, protein complex

  • and we'll pick them out and generate galleries,

  • like this, that show our different molecules.

  • These are showing the molecule in different in-plane orientations, but also different views.

  • So, what we do computationally is we go through a process of aligning these images to each other

  • and then classifying them.

  • So, eventually we put everything that shows the same view in different classes

  • and now these are ready for averaging

  • and the averaging will give us now enhanced views of each of these orientations of the molecule.

  • These now have to be related to one another

  • and this is a very tricky step that I'm completely going to forget about for now,

  • but ultimately this can be very computationally involved

  • but ultimately if the relative orientations of these different views are obtained,

  • we can go and reconstruct the object in 3 dimensions.

  • Let me now illustrate how do we go from the 2 dimensional images

  • that we know are related to one another by defined angles to obtaining the 3 dimensional reconstruction.

  • We do that by something that is called back projection.

  • So, imagine now, this is a very simple example, where your object, your molecule, is made up by these three circles.

  • So, when you pass electrons through it, you generate a 2 dimensional projection that

  • looks say like this. And of course, you're going to have this object in different orientations in your EM grid,

  • which means that when you take different images,

  • what you get is different projections that look distinct

  • and that by some means you're able to place

  • one in relation to each other by finding the relative angles.

  • This is tricky to do, but once you've done it, the way to obtain the reconstruction is to back-project.

  • What does that mean? You take each one of these projections

  • and you smear it and you see how all of them intersect,

  • reproducing the object.

  • So, I have another movie that is a little bit more fanciful

  • because St. Patrick's day is coming, the day that we are filming,

  • so this is our object and what I want to illustrate here is

  • how as we use more projections, that are equally distributed,

  • we get more and more accurate representation of the object.

  • So, this is a movie in which now projections are being added and the intersection is giving rise to this leaf now

  • in more and more detail.

  • Let me now illustrate all of these with a real project.

  • This is our study of the exosome,

  • which is a molecular machine that is involved in processing RNA

  • and in some cases degrading RNA.

  • And the exosome, in this case from yeast, from budding yeast,

  • was purified and each one of these lumps that you see correspond to one complex, one image of the complex.

  • And the complex is randomly oriented in here,

  • and this is actually, by the way, a negative stain image, so all that I showed you up to now was cryo-EM,

  • but this is an example of a negative stain study.

  • So, if you go and pick out individual particles, this is how they look like,

  • this is a gallery. They're pretty noisy,

  • but going through the process of alignment and classification, averaging,

  • you get now images like these, that look a lot more well-defined.

  • So, the tricky part, which I'm skipping, is how each one of these images are related to one another,

  • but ones that were sorted, we were able to obtain a 3-dimensional reconstruction.

  • Just to tell you, we obtained two reconstructions,

  • one of the full complex, that is shown here

  • and one of a core element in the complex,

  • whose structure had been obtained at atomic resolution by X-ray crystallography

  • of the human homologue.

  • When we subtract one from the other, we get the core in blue and this extra region in yellow,

  • which happened to be the one that has the biochemical activity,

  • the site that actually chops the RNA.

  • Now, what is shown here is now the crystal structure of the human homologue

  • of the core domain. And what you see here in yellow are pieces that were taken from homologues found via bioinformatics.

  • And what this allowed us to do was to create a pseudo-atomic model

  • of how the top and the bottom part of this structure interact.

  • Now, this is actually a very common type of methodology.

  • We refer to this as hybrid methods

  • and it involves the docking of crystal structures of components

  • into the low-resolution structure of the full, functional complex.

  • And this is something that not only tells you how good your structure is,

  • but also gives you new information on,

  • say, interfaces, how this bottom part, and the top and bottom part, interact,

  • which elements are involved in that interaction.

  • And in this particular case, it gave us the path of the RNA by aligning the cavity

  • in the top part with cavities that exist in the active region

  • that lead you all the way to the active site.

  • So, no matter whether the molecule that we were studying was in a 2-dimensional crystal,

  • in a helix, or was a single particle with many copies,

  • we're utilizing and combining to generate the structure,

  • we were always looking at things where there were many copies,

  • of, identical copies of an object.

  • But what happens when we're interested in something where no two are the same?

  • Like, when we're interested in organelles or cells. What do we do here?

  • In that case, what we utilize is the method of electron tomography.

  • In electron tomography, the basic principle is all the views that are required to obtain a reconstruction

  • have to be taken from a single object.

  • Not from identical copies, but from a single object.

  • So, in here, again, the idea is we have a very unique sample for which there's no identical one,

  • say, an organelle or a piece of a cell.

  • And what we're going to do is we're going to take many views of the object.

  • By taking this object and tilting it,

  • and always looking and shooting,

  • grabbing images from the same object.

  • The images will be obtained by tilting very gradually, typically about 1 degree,

  • although how fine that division is made depends on the size of the object.

  • In here, how these images are related to one another is very easy.

  • It's just determined by you; you were always looking at the same object

  • and you were the one telling the microscope to tilt by a certain degree.

  • So, computationally, experimentally and computationally, it's very easy to obtain a reconstruction,

  • which in this case is again done by back projection.

  • The difficulty here, as you will see, has to do with interpreting these images

  • which seem to be noisier and are of organelles that are really, really very complex.

  • So, we have utilized this kind of methodology in my lab

  • to study yet another self-assembly system

  • and that is the one of septins, which are proteins that self-assemble and make

  • filaments that actually line particular sites near the membrane in the cell

  • at the position where cell division is going to take place.

  • We study septins in the organism where it was first discovered, which is the budding yeast.

  • What we're interested in when we look at these cells is just the particular region here,

  • where a filament has formed and where we want to see how they're organized and how they interacting with the cell membrane.

  • So, the first thing that we do is collect a tomographic tilt series

  • where we place the object in the electron microscope, decide what it is we're going to shoot at

  • and then take images, once for every tilt of one degree.

  • And here, these images are showing just one right after the other,

  • so these don't correspond to a reconstruction yet, this just shows you one after the other the images of a very thick section

  • where it is very, very hard to determine what is in there.

  • So, after this tilt series are used in back projection, we can generate the reconstruction

  • and I'm going to show it to you as a series of slices

  • going in and out several times in the reconstructed section.

  • So, this is the section, we started at the edge of the section and now we're going through

  • and I hope that you can see now that we see in much detail as we're going

  • each one of these speckles corresponds to ribosomes

  • and there's many of them in the cell, what you see here is the double membrane of the nucleus.

  • There's a lot more endomembrane here and of course,

  • right by the edges is where we're going to see our object of interest,

  • which are the self-assembly of septins into filaments

  • around the membrane.

  • So, you can see the complexity of reconstructions like this,

  • there's so much going on. So, in order to be able to look at it at once

  • what we do is we simplified this image, but just utilizing simple surfaces and lines

  • to trace through the surfaces of the plasma membrane,

  • of the nuclear membrane, of the filaments

  • that we can trace from one section to the other.

  • And we get a rendition like this by what we call segmentation.

  • So, this is now a very simplified view where we eliminate the things that we were not interested in,

  • like all of the ribosomes. And what you see here in yellow is the plasma membrane,

  • it's very curved because that's site of the bottleneck where the septation,

  • the division of the mother and daughter cell, is going to take place.

  • This section included a nucleus that is also in the process of dividing

  • with microtubules shown here in red that are pulling chromosomes apart.

  • There is more membrane that is internal that is shown here in kind of orange.

  • Actually, the thing that we were interested in looking at are these filaments

  • that run in a number of directions, they run around the circle

  • if you want, in the bottleneck, but they also run between daughter and sister cells

  • so, they're the ones that we're showing here in green,

  • the ones that we're showing in blue

  • and interestingly, there are also small filaments that are shown in red

  • that are connecting the membrane to this filament system.

  • So, just as a final note, imagine all the information that is contained in the tomogram that I just showed you a minute ago

  • where we only concentrated on this small section.

  • It would be great if tomograms are made available, publically available,

  • just like crystal structures or electron maps of reconstructions

  • so that anybody, irrespective of what you work on,

  • can take, can make use of the image to follow and track

  • the object of their principle interest.

  • So, this is the introduction that I wanted to give you of this technique

  • and I hope that in this brief time I gave you an idea of the generality of how applicable this method can be,

  • all the way from individual molecules to visualization of the cell.

  • And what I haven't had any time to tell you that this method is far from being totally optimized

  • and that there is a lot of development and improvement in the pipeline

  • that is going to allow us to get not only higher resolution,

  • but study even more systems that right now remain really challenging.

  • So, by the time someone like you is ready to use this technique, things will have really moved beyond

  • what I showed you today. So, I really cannot wait for that moment myself.

Hi, my name is Eva Nogales, I'm a professor of molecular and cell biology at UC-Berkeley,

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