6 - Cryo-EM and the Chaperonin Mechanism

Question 1

What are the benefits and limitations of fluorescence microscopy?

Answer 1

Fluorescence microscopy:

• Can be used for tracking
• Can be used to characterize general structure at molecular level
• Can’t be used for seeing details within the cell

Question 2

What kind of image does Cellular cryo-electron tomography provide?

Answer 2

Cellular cryo-electron tomography allows you to get a 3D picture of what’s going on in the cell. The sample must be thin in order to carry this out. This allows you can see the outline of the cell membrane, vesicles, filaments.

Question 3

How can fluorescence and Cryo-em be combined?

Answer 3

There are now methods to track where fluorescence was (in fluorescence microscopy) on the electron microscopy of the cells, to view movements on a cellular level.

Question 4

What uses does Cryo-EM tomography have outside cellular imaging?

Answer 4

Cryo-electron tomography can also be carried out on a smaller scale, e.g. to look at virus structure or the structure of macrocomplexes such as GroEL/GroES or microtubules. This produced high-resolution 3D views of samples in terms of their electron density.

Question 5

What is Sub-tomogram averaging?

Answer 5

In Sub-tomogram averaging, multiple copies of the object of interest are then extracted from the tomogram, aligned and averaged in 3D to generate a higher-resolution reconstruction. For example, this allows you to cut out bits of the spikes on a virus, which aren’t organised in a regular way, and use this to get an average structure.

Question 6

What can be produced from Single particle cryo-EM?

Answer 6

This is a way of looking at the structure of macromolecules without needing crystallisation or isotopic labelling. It used to be restricted to fairly low resolution, resulting in information about the general shape but not the internal details, but resolution has improved and it is now possible to get atomic-level structural detail. This is an advantage as crystallisation is hard for very large complexes.

Question 7

What is TEM?

Answer 7

In transmission electron microscopy, the electron beam goes through the sample and thus the sample must be thin.

A TEM image is formed due to the thin specimen scattering electrons. Interference between scattered and unscattered electrons gives phase contrast image, which is the 2D projection of the original 3D object- not a cross-section.

Question 8

How can TEM be used to produce 3D structures?

Answer 8

2D crystals can be assessed using electron crystallography viewing at different tilts, this produces an electron diffraction pattern.

Interference between scattered and unscattered electrons gives phase contrast to image.

Image is 2D projection of original 3D object, so the 3D structure can be determined from a set of views at different orientations.

Question 9

What can TEM be used on?

Answer 9

Electron diffraction of microcrystals, which are smaller than 1μm (same principles as x-ray crystallography, except x-ray requires bigger crystals)
Whole cells or organelles (tomography of unique objects, cumulative irradiation).
Icosahedral viruses. These are symmetrical, with a 60 fold axis of symmetry. These were the first structures for which atomic structure could be obtained by TEM, allowed by the symmetry.
Helical assemblies
Asymmetric single particles, such as the ribosome. A reconstruction of an asymmetric molecule is possible but requires a lot of data.

Question 10

What is the limitation of TEM analysis of whole cells?

Answer 10

You can’t do averaging on whole cells because cells are not identical: you can only average bits inside them that may be repeated. TEM allows you to see quite a lot of cell structure.

Question 11

What is the major limitation of TEM?

Answer 11

The ultimate limit on resolution is damage to the specimen by the electron beam, and consequently it is important to be careful with the exposure of the electron beam onto the molecule.

The sample is tilted in cryo-EM, and images taken at these different tilts, resulting in further damage by the electron beam.

Question 12

What gives TEM such great versatility?

Answer 12

In the conventional TEM we have the option of magnifying the image of the sample formed by the objective lens, or the diffraction pattern.

The ease with which the microscopist can move between the two modes (imaging mode and diffraction mode) is one of the things which makes the TEM such a useful and versatile instrument.

Question 13

How state must the sample be in for Cryo-EM?

Answer 13

Because electrons interact strongly with matter, the electron path of the microscope must be kept under high vacuum to avoid unwanted scattering by gas molecules in the electron path and thus the specimen must be in the solid state (i.e. frozen). Special preparation states are necessary to either dehydrate or stabilise hydrated biological samples under vacuum.

Question 14

What is negative staining?

Answer 14

It is called negative staining as you see the heavy metal stain outlining the molecules, rather than seeing the molecules themselves. It is seen by exclusion rather than the binding of the stain.

This provides information about the size, shape and symmetry of the particle, and an overview of the homogeneity of the preparation.

Question 15

What are the advantages of negative staining?

Answer 15

It involves a simple procedure, it is quick to check samples and is high contrast. Additionally, it can be done for small structures, less than ~100-200kDa, for which the signal in cryo-EM may be too weak for accurate detection and orientation determination.

Question 16

What is the procedure for carrying out a negative stain?

Answer 16

On a carbon support screen, supported by a copper mesh, put a droplet of the sample protein in buffer onto the grid, allow it to spread out onto the grid.

Next, add the heavy metal stain and blot again. Leave to dry, resulting on pools of dried-down molecules.

The heavy metal is deposited as a dense coat and outlines the surfaces of the biological assembly.

Question 17

What stains are often used in negative staining?

Answer 17

The most common heavy metal salt solution used is uranyl acetate, which gives the highest contrast. However some samples are better preserved in other stains, e.g. tungsten salts.

Question 18

What are the disadvantages of using heavy metal salts in negative staining?

Answer 18

The use of heavy metal salts and dehydration of molecules leads to possible distortion and flattening. The stain may not cover the entire molecule, so parts of the structure may be distorted or absent from image data.

Question 19

What are the advantages of the preparation used for cryo-EM over negative staining?

Answer 19

it allows imaging of the molecules in their native, hydrated state at near physiological conditions, as the samples are frozen so quickly that the water becomes vitreous instead of crystalling.

Macromolecules and cells are usually in aqueous solution and hydration is necessary for their structural integrity so the 3D structure is preserved and the rapid freezing is capable of trapping transient states.

Additionally, the low temperature slows the effects of electron beam damage.

Question 20

What are the disadvantages of Cryo-EM preparation techniques compared to negative staining?

Answer 20

Cryo EM involves a more complex preparation, longer time for checking samples, and results in a low contrast in the image, as there is no staining.

Question 21

How are samples prepared for cryo-EM?

Answer 21

The sample is put onto the EM grid and then blotted before the grid is plunged into liquid ethane.

Question 22

What is used for the grid in cryo-em?

Answer 22

The support may be carbon or, as this contributes to additional background scattering, may be perforated films, in which the sample is imaged in regions of ice suspended over holes in a support film.

Question 23

Why are the samples frozen in liquid ethane instead of nitrogen?

Answer 23

Freezing in liquid nitrogen is very slow, due to a nitrogen gas layer which forms and insulates it. When things cool slowly, the water crystallises and the structure can become distorted.

When freezing in liquid ethane the water remains as a liquid and transfers the energy out of the sample much faster, resulting in a vitrious water layer. This stops the molecules in their tracks in their native, hydrated state, as they are at room temperature, even though they are frozen. It prevents the formation of ice crystals which would be damaging to the specimen.

Question 24

What sort of image is produced by Cryo-EM?

Answer 24

The resulting image looks weak and grainy, although the protein has a slightly darker line outlining it. This is because there was a low electron beam used to avoid frying the molecules.

Additionally, the contrast is also low due to the intrinsic low contrast between macromolecules and water: there is intrinsically very little difference.

In order to get good resolutions, single-particle cryo-EM techniques must be used.

Question 25

What can single-particle EM be used on?

Answer 25

Single particle cryo-EM can be carried out on isolated large macromolecular complexes, greater than 100-200kDa.

These need to be randomly orientated in solution, and can be trapped in different reaction states by vitrification, e.g. if a conformational change takes place once the protein binds ATP.

Unlike in X-ray crystallography, no crystallisation or ordered assembly is needed.

Question 26

What must be determined about the structures in single particle cryo-EM for 3D reconstruction?

Answer 26

Whe position and orientation of each particle must be determined (whereas in XC once you have determined this for one you have determined it for all). Consequently, long computation is needed.

Question 27

What determines the resolution of single particle cryo-EM?

Answer 27

The more particles used, the higher the resolution, up to 3Aͦ. However, as previously mentioned, the ultimate limit is radiation damage.

Question 28

What is ‘purification in the computer’?

Answer 28

In single particle cryo-EM mixed states can sometimes be separated in single particle analysis. Although long computational sorting is needed, you can get multiple structures from the same data set, as you can get sets of images from particles which are not the same.

Interpretation can be done by atomic structure docking or direct determination of backbone

Question 29

How can the signal:niose ratio be improved in single particle cryo-EM?

Answer 29

The signal:noise ratio can be improved by averaging similar views. The individual raw images aren’t that great, with low contrast. A lot of data must be collected to get a lot of orientations in order to get a 3D structure, but this also allows you to do a lot of averaging.

As you average signals more and more, if the signal is the same, the image will get better and better, averaging out the noise.

Question 30

What is ‘particle picking’ in single particle cryo-EM?

Answer 30

Individual particles are identified by shape.
Then, computational/statistical methods are used to classify the objects into groups based on objects that look like each other, separating them from the ones that look different to determine which can be used to average each other.

Question 31

How can ‘particle picking’/class determination of a single particle cryo-EM image be refined?

Answer 31

Averages of the classes gives you more information, which you can then use in alignment.

Initial class averages selected from the first round of classification can serve as references to bring similar images to the same in-plane position and orientation and to separate different out-of-plane views.

Alignment is done by finding shifts and rotations that bring each image into register with a reference image. These are repeated so that successive averages contain finer details, in term improving the reference image, for subsequent rounds of refinement.

Question 32

How high resolution is single particle cryo EM?

Answer 32

Due to improvements in resolution, single particle cryo EM can be used to obtain the same results as x-ray crystallography: atomic resolution. For example, Bartesaghi et al (2015) obtained a 2.2 Aͦ resolution cryo-EM structure of β-galactosidase.

Question 33

How are 3D structures produced by electron tomography?

Answer 33

Electron beams directed at the 3D object result in a set of 2D projections. The 2D projections of various molecular orientations are back projected to reconstruct the 3D structure.

Didn’t you already ask this?

Question 34

What limits the resolution of electron tomography?

Answer 34

When the electron beam hits the samples, due to the electrons being highly energetic the sample moves a bit, thus blurring it the image.

Question 35

How is the decrease in resolution in electron tomography due to motion of the target solved?

Answer 35

Direct electron detectors are high speed and record images as movies. Consequently you can correct for the motion by aligning the movie sub-frames.

These new detectors allow you to obtain a higher resolution image as they effectively allow you to get back the data which is lost due to movement.

Question 36

How do chaperonins select misfolded proteins?

Answer 36

They have hydrophobic sites to trap proteins that are in the non-native state.

Question 37

What is he structure of GroEL?

Answer 37

E. coli GroEL is a huge, 14-mer complex with two states. These proteins are arranged in two heptameric protein rings that are lined on the inside with hydrophilic residues.

Question 38

What is required for GroEL to fold a protein and what does this show?

Answer 38

ATP binding and GroES is all that is required for the protein to fold. Thus, the chaperonins assist folding without imparting steric information.

Question 39

How does ATP bind to GroEL?

Answer 39

ATP binds with positive cooperativity to one ring but reduced cooperativity between rings, resulting in an altered conformation with reduced substrate affinity.

Question 40

What happend when the ATP binds to GroEL when a protein is inside?

Answer 40

The ATP-bound ring rapidly binds GroES (lid), simultaneously sequestering the hydrophobic binding sites and encapsulating the substrate in the folding chamber with a hydrophilic lining. There is a massive conformational change in the GroES-bound ring.

Question 41

How does the GroEL/GroES complex assist in protein folding, and what happens after it does?

Answer 41

Substrate folds (or doesn’t fold!) inside the chamber due to the lack of exposed hydrophobic sites or other partners for aggregation, along with limited enclosure site.

The ATP bound to the ring is hydrolysed, allowing ATP binding to the opposite ring that primes the release of GroES and the trapped substrate.

From here the cycle can repeat.

Question 42

How does bacteriophage T4 capsid protein gp23 interact with the GroEL/GroES complex?

Answer 42

gp23 is a 56 kDa protein which requires the full chaperonin complex (~900kDa) for its folding. It is at the upper size limit of what can be folded in GroEL.

Because of this it was used for imaging of GroEL-substrate complexes.

Question 43

How were permanent GroEL-gp23 complexes formed?

Answer 43

Gp23 was denatured in 6M urea and then diluted into a GroEL-containing buffer. The full chaperonin cage was formed by adding the T4 GroES analogue gp31 and a non-hydrolysable ATP analogue.

Heterogeneous complexes of the chaperonin and substrate formed. These needed to be sorted into more homogeneous classes for structure determination, without imposing any symmetry.

Question 44

What technique can be used for imaging heterogenous complexes?

Answer 44

Eigenimage analysis

Question 45

What is Eigenimage analysis?

Answer 45

This allows you to sort out the main components of variation in your data set, eg proteins binding to the complex in different places.

Once you have the principle component of variation, you can use this to separate out the molecules into classes that have it and those that do not.

Question 46

What did Eigenimage analysis of GroEL-gp23 complexes show?

Answer 46

Most of the ternary complexes formed fell into three structural classes: ‘empty’, with no apparent substrate density, substrate bound in the trans (open) ring, and substrate bound in both cis and trans rings

Question 47

What did the structures of the GroEL-gp23 complexes imply about the mechanism of the chaperonin?

Answer 47

The major domain of gp23 is wedged into the chamber in a restricted position where it folds. The small insertion domain, which makes inter-subunit contacts in the viral capsid, is still disordered and probably folds when it the capsid hexamers are assembled.

The encapsulated gp23 substrate causes an expansion of the folding chamber, distorting the cage.

Thus, this has shown that folding substrates directly and indirectly affect the conformation of the chamber.

Question 48

What did Cryo-EM and atomic structure fitting of GroEL-ATP complexes allow visualisation of?

Answer 48

the conformational changes that occur upon ATP-binding (Clare et al 2012).

Question 49

How were the different conformations of GroEL captured for imaging?

Answer 49

3D reconstructions were produced for the apo state, three GroEL-ATP states with ATP bound in one ring and three with ATP bound in both rings.

Question 50

What were the major conformation changes demonstrated for the GroEL structure in the different ATP binding states?

Answer 50

The major movements are a large rotation of intermediate and apical domains forming the Rs and Rd states followed by elevation and radical expansion of the apical domains to the R-open states.