Lecture 4: Imaging Viruses

Question 1

Describe the light microscopy technique

Answer 1

This technique can image anything larger than the shortest wavelength of visible light. It is good for cells but generally useless for viruses because most viruses are smaller than the shortest wavelength of visible light (except for the very largest giant viruses).

Question 2

Describe the electron microscopy technique

Answer 2

EM has higher resolution because energetic e- have shorter λ (DeBroglie).
So EM can image viruses.
Flavors of EM include:
-Freeze- etch EM (imaging membrane surfaces)
-Cryo-EM w/ 3D reconstruction from 2D images

Question 3

What are two other imaging methods?

Answer 3

Atomic force microscopy and X-ray crystallography

Question 4

How does Electron Microscopy work?

Answer 4

In EM e- are accelerated & focused (via magnetic, not glass, lenses) and lenses magnified images are viewed on fluorescent screens (where e- converted to light) then either photographed (old) or CCD camera to image either visible light or e- directly (modern EM).

Question 5

What is the resolution limit for the light microscope?

Answer 5

~3000 Å resolution limit

Question 6

What is the resolution limit for the transmission electron microscope?

Answer 6

25-50 Å resolution limit for stained viruses depending on type of stain used.

Question 7

What is TEM?

Answer 7

This is standard EM technology (transmission electron microscopy)

Question 8

What types of viruses were initially imaged?

Answer 8

In the 1950s initially bacterial viruses were imaged because they were hard and robust and could survive e- bombardment.

Question 9

What helped contribute to the small pox vaccine?

Answer 9

It was discovered that heavy metal staining can be used to protect the virus when imaging.

Question 10

What other types of viruses were imaged in the 1960s?

Answer 10

Animal viruses (that were smaller and softer).

Question 11

What are two challenges in doing EM?

Answer 11

Because e- has short λ = higher energy (amount of radiation required to collect image is comparable to placing sample ~20 m away from a thermonuclear device) leads to sample penetration by e-. Biological samples are particularly highly penetrable to electrons:
1) Eventual sample damage (e- radiation damage)
2) Low image contrast (contrast = diff between reflected and transmitted light) (Hence glass is invisible)

Question 12

What are the solutions to the two challenges of doing EM?

Answer 12

To limit penetration (more limited damage, higher contrast) you can coat (stain) sample with heavy metal (Pt, Os, Mo, U)
-Positive staining
-Negative staining

Question 13

Describe positive staining

Answer 13

(dark viruses on light background). Metal chemically attached to proteins (in the virion) and increases their density wrt background. Stained (dense) areas appear occluded (dark) on fluorescent screen.

Question 14

Describe negative staining

Answer 14

(light viruses on dark background): coating covers mainly intervening spaces between virons: virions stick up through stain, are therefore covered with less thick stain than the immediately surrounding ring or meniscus (surface tension) so virus appears lighter and is surrounded by a ring of dark stain.

Question 15

What is an issue with the staining technique?

Answer 15

Staining conditions (sample preparation) are now harsh on the sample (i.e. sample fixation with alcohol, chemical reaction with stain at low pH) sample immobilization in EM, dehydration through placement in high vacuum of EM. This is even before it gets bombarded with thermo-nuclear amounts of electrons.
Also possible structural collapse so you don’t know whether images represent true functional forms of the virus.

Question 16

What is one type of EM that is popular and an good alternative to the normal negative and positive staining techniques?

Answer 16

Cyro-EM

Question 17

How does Cryo-EM work?

Answer 17

Virions are first trapped in non-crystalline (vitreous) ice (V.I lacks destructive ice crystals) This ice never dehydrates in EM vacuum (cuz sample in EM is kept below sublimation temp/pressure of ice) so sample also never dehydrates.
So: no staining or fixing is required (native environment for proteins). Avoids structural deformation due to stain or ice crystals. Image relies purely on electron scattering by unstained virus protein/DNA/lipid.
Also Cryo-EM uses low doses of electrons to prevent radiation damage to an unstained sample (ice also protects from radiation).

Question 18

What is an issue with Cryo-EM?

Answer 18

Low e- dose + no stain = faint signal noise = low contrast image

Question 19

What is the solution to the low contrast image in Cryo-EM?

Answer 19

Computational image enhancement (de-noising) by averaging of multiple comparable images. Each vision was frozen in the ice in slightly different (random) orientation, find the subset of images that look the same = the same orientation then sum them to higher contrast = image “class”
Then do computational 3D image reconstruction:
Each image class represents a different virus orientation.
-Computer tomography algorithm to reconstruct a 3D composite of the thousands of the individual 2D rotational image classes (exactly as in a CAT scan).
-In fact, during the EM, the sample may be progressively tilted as in a CAT scan, to verify the 3D reconstruction.

Question 20

What is one example of Cryo-EM?

Answer 20

computer enhanced 3D reconstruction image reconstruction of capsid of HSV-1

Question 21

How does atomic force microscopy work?

Answer 21

1) Incredibly sharp needle rapidly taps a surface.
2) Any virus on a flat surface transiently deflects tip up a bit during tapping (tip follows topography of sample).
3) Laser interferometer measures height of needle with exquisite sensitivity/spatial resolution (to within half wavelength of laser’s light beam).
4) Image reconstructed from the transient changes in height.

Question 22

How does laser interferometry work?

Answer 22

Deflect a mirror just half a wavelength and the two signals go from reinforcing to canceling.

Question 23

What are the strengths of atomic force microscopy?

Answer 23

-Image shows individual virions (not an average of many individuals): See variation.
-Imaging done in air or under physiological liquid (no sample prep perfectly gentle).
-Can make movies (e.g. of virus entering cell) if it can scan each frame fast enough)!

Question 24

What are the weaknesses of atomic force microscopy?

Answer 24

AFM only images the surface only (non-penetrating).
It can also damage samples if the dose is too high

Question 25

How easy is it to image virions of pox viruses?

Answer 25

Virion of Pox are large, complex, with irregular shapes and can be difficult to characterize.

Question 26

Virion of Pox by EM results

Answer 26

Textbook images of Vaccinia virus by EM with various staining methods prove to be inconsistent and crappy.

Question 27

Virion of Pox by AFM results

Answer 27

Topographical views of Vaccina by AFM is much more consistent

Question 28

Describe how AFM compares to EM imaging.

Answer 28

-No staining
-No acid
- No drying
-No vacuum
- No e- bombardment
- No shrinkage
Surfaces are imaged as they really are.

Question 29

Other than imaging what else is EM and AFM used for?

Answer 29

Both techniques are also one of the only ways to count virus particles.
Count within just a tiny volume (just a fraction of 1 mL) of sample (back-calculate particles/ mL of original sample).

Question 30

Why is it important to understand the number of virus particles?

Answer 30

Infectivity (ratio of total # of particles: # of infectious particles)

Question 31

What is the highest resolution imaging technique of all?

Answer 31

X-ray crystallography

Question 32

What are the strengths of X-ray crystallography?

Answer 32

-Very high resolution of all images
(atom-scale)
-Internal structures (inside virus) may be resolved.
-Some of the smaller, regular-shaped, non-enveloped (naked capsid) viruses (e.g. the picornaviruses and some plant viruses) are amenable to X-ray crystallography.

Question 33

What are the weaknesses to X-ray crystallography?

Answer 33

Slow and difficult to process data.
Have to make array for crystal.
Can take years to get a good image.

Question 34

Describe an example of the icosahedral virus x-ray crystal structure and symmetry of capsid of Desmodium Yellow Mottle Virus of plants

Answer 34

-Assembles from 180 single protein molecules (9 per icosahedral face).
- Strands represent the backbone of capsid polypeptides. In order to recognize symmetry in the capsid we have stripped away (from the image) sidencahins of the capsid protein and the genome.
-Can be viewed as 12 overlapping pentameric capsomers (white).
Pentameric Capsomers illustrate one of the icosahedral symmetry requirements namely 5 fold axis about each vertex.
Can also see 3 fold axis at the center of each face.
Also blue, red, yellow = same protein sequence from same gene.
But
An individual protein molecule always has an asymmetric 3D shape (no symmetry, 180 asymmetric shape = 1 icosehdron).

Question 35

What type of shape does an individual protein molecule have?

Answer 35

An individual protein molecule always has an asymmetric 3D shape (no symmetry, 180 asymmetric shape = 1 icosehdron).

Question 36

What is a mathematical fact regarding icosedrons?

Answer 36

Within the triangular face of any icosehdron it is impossible to arrange >3 identical asymmetry shapes into symmetrically identical environments.
Ex: If there were only 3 identical asymmetrical molecules per triangular face, each one would indeed be in an identical environment.
You can build an icosedron from 60 perfectly identical molecules of A.
3 per triangular face though A is asymmetric in itself (multiplied by 20 faces).

Question 37

A capsid has 9 molecules per face. How do we fix this problem?

Answer 37

9>3 asymmetric shapes in a face.
We need to reduce it to three clusters of three.
Each cluseter comprises of red- blue-yellow trimers.
There are 3 such trimers per face

Question 38

Nature builds __ to ensure that there are only three __ asymmetric shapes per icosahedron.

Answer 38

1) multimers
2) multimeric

Question 39

How many protein molecules are now asymmetrical ABCD tetramers.

Answer 39

12 protein molecules in the pink face now are three asymmetrical ABCD tetramers.

Question 40

Describe the example of the multimer

Answer 40

The C/A face always docks to A/B.
The A/B face always docks to C/A
The left side of D always docks to half of B
The other two sides of D always dock to D.
3 asymmetric shapes and each are in an identical environment.

Question 41

ABCD have to be ___ different

Answer 41

subtly to acordate their distinct environments.
“quasi-equivalent structures”

Question 42

What is the main point being the icosahedral virus?

Answer 42

Blue, red, yellow = same protein sequence from same gene but within the BRY trimer these three protein chains have only quasi-equivalent structures.

Question 43

What are the pros and cons of EM?

Answer 43

1) Can be used for staining.
2) Okay radiation damage (metal stain protects).
3) Okay resolution (can see individual protein complexes).
4)Averaging: No (individual particles).
5) Can view interior of virus. (Only if first to make slices through sample eg freeze etch.)

Question 44

What are the pros and cons of Cryo- EM

Answer 44

1) No staining
2) May or may not have radiation damage at low dosage however the ice protects
3) Very good resolution (protein backbone, sometimes side chains too).
4) yes averaging
5) yes if interior is not randomly disordered.

Question 45

What are the pros and cons of AFM?

Answer 45

1) No staining
2) No radiation damage
3) Ok resolution of protein complexes.
4) No individual particles
5) No view of the interior virus

Question 46

What are the pros and cons of X-ray crystallography?

Answer 46

1) No staining
2) May or may not have radiation damage (X-rays damage sometimes).
3) Yes resolution (excellent individual atoms).
4) Yes averaging
5) Yes view of interior virus is seen if interior is not randomly disordered.

Question 47

What are the main key points of lecture 4?

Answer 47

• Anything smaller than the wavelength of light, cannot be imaged by light microscopy.
  -Other imaging methods (EM?cryo EM, AFM, X-ray crystallography) each have strengths and weaknesses
• EM good for viruses which are generally smaller than the wavelength of light.
  -EM requires staining which is destructive, Electron bombardment is also destructive.
  -EM does not easily give height info, so 3D imaging is tough.
• Cryo EM helps solve the destruction problem by freezing virus in ice.
• Cryo EM may also get around 3D problem, by allowing images at different orientations followed by computer reconstruction of a 3D image.
• But Cryo EM averages many particles into one image.
  AFM is a very gentle surface imaging technique which is good for viruses that are essentially destroyed during EM preparation or EM itself. Poxviruses and their innards (which can be revealed by removing surface layers).
  -AFM does not have any of the above problems (for surface imaging).
  -EM and AFM images allow us to count virions
  X-ray crystallography reveals atomic detail and has been successful for several viruses. It shows the symmetry of icosahedral capsids.
  -Quasi-equivalence the same protein repeated many times in an icosahedral capsid but there may be a subtle change in tertiary structure between repeating units.