Near-Atomic Resolution Achieved Using - PowerPoint Presentation

Slide1

Near-Atomic Resolution Achieved Using Cryo-EM

Lindsey

Organtini

8-16-13

Structure Work GroupSlide2

Virus assembly

Structure is a key element in understanding viral assembly

X-Ray crystallography can resolve atomic structural information, so why not use it?

Stringent requirements for crystallization not suitable for all functional states

Can suspend particles in specific state using vitreous iceSlide3

Viruses in CryoEM

Particularly suited for

c

ryoEM

due to their high symmetry, molecular mass, stability, and solubility in buffers

Have been used since the inception of

cryoEM

De Rosier and Klug used T4 bacteriophage tails in their 1970 paperSlide4

Fun Fact

As of 2010, ~20% of all entries have achieved resolutions better than 10Å!Slide5

The Importance of Resolution

Improving resolution means more structural features are discernible

Low resolutions (20-10 Å)= general shape,

capsomere

morphology

High Resolution (9-6 Å) = individual subunit boundaries, secondary structure elements (

α

helices,

β

sheets)

Near Atomic Resolution (<

4.5

Å) = Pitch of helices, separation of

β

strands, some side chains of

a.a

.

Able to determine features unable to be crystallized

Can use both in conjunction in order to learn moreSlide6

Why resolution matters…

Not near atomic, but improved resolution can show make a big difference in interpretation!Slide7

N-termini of EV71 not resolved in crystal structureSlide8

We’ve seen what high resolution can achieve, but what about near-atomic resolution?Slide9

Ribsome

details with increasing resolutionSlide10

At 3.8Å, see helices and sheets in Rotavirus VP6Slide11

New Discoveries in ε15 Phage

Previous reconstruction 9.5Å -> Added 20,000 more particles to achieve 4.5

Å

gp7

gp10Slide12

Little sequence but high structural similiarities

CryoEM

shows subtle differences between the three structuresSlide13

31,815 particles used to achieve 3.6Å of 2 major proteins (

hexon-trimers

and penton base)

Reveals N terminal arm not resolvable in X-ray

Similar to arm of rotavirus, which was also revealed by

cryoEM

and unresolvable in X-ray

Advances in

AdenovirusSlide14

Minor proteins in Adenovirus

Used to attach major proteins onto lattice

3 proteins resolved high enough to model

Able to detect side chains

X-ray could only resolve 2 proteins partiallySlide15

P22 captured in multiple conformational states

Provirion

3.8Å with 23,4000 particles

Virus

4.0 Å with18,3000 particles

Virion

is 100

Å

wider and more angular than

provirion

Hexamers

skewed in

provirion

which become more symmetric in

virionSlide16

P22 captured in multiple conformational states

Cyan =

procapsid

Magenta =

virionSlide17

P22 captured in multiple conformational states

Provirion

3.8Å with 23,4000 particles

Virus

4.0 Å with18,3000 particles

Virion

is 100

Å

wider and more angular than

provirion

Hexamers

skewed in

provirion

which become more symmetric in

virionSlide18

P22 captured in multiple conformational states

Procapsid

Virion

Between

capsomeres

Between asymmetric unitsSlide19

So how do you achieve near-atomic resolution?

Use many, many particles (10x what is normally used)

Automated data collection

Will need the computer resources

High quality images

No lens aberration or drift

CCDs cause information lose

Improved defocus measurements and avoiding alignment error . . .Slide20
Slide21

There is still a place for x-ray crystallography!Slide22

CryoEM + X-ray

Combination of both for pseudo atomic resolution

Pseudo Atomic Modeling Example (Virus +

FAb

)

Fragment of

AbSlide23
Slide24

Imagine the information we could achieve by combining near-atomic resolution cryoEM

and x-ray data!