NEAR-ATOMIC RESOLUTION
ACHIEVED USING CRYO-EM
Lindsey Organtini
8-16-13
Structure Work Group
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 ice

VIRUSES IN CRYOEM
Particularly suited for cryoEM 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
paper
FUN FACT

As of 2010, ~20% of all entries have achieved resolutions better
than 10Å!
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 more

WHY RESOLUTION MATTERS…
Not near atomic, but improved resolution can show make a big difference in interpretation!
N-TERMINI OF EV71 NOT RESOLVED IN CRYSTAL
STRUCTURE
WE’VE SEEN WHAT HIGH RESOLUTION CAN ACHIEVE, BUT
WHAT ABOUT NEAR-ATOMIC RESOLUTION?
RIBSOME DETAILS WITH INCREASING
RESOLUTION
AT 3.8Å, SEE HELICES AND SHEETS IN
ROTAVIRUS VP6
NEW DISCOVERIES IN Ε15 PHAGE
gp7
gp10
Previous reconstruction 9.5Å -> Added 20,000 more
particles to achieve 4.5Å
LITTLE SEQUENCE BUT HIGH STRUCTURAL
SIMILIARITIES
CryoEM shows
subtle
differences
between the
three structures
ADVANCES IN
ADENOVIRUS
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
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
partially
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 virion
P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES
Cyan = procapsid
Magenta = virion
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 virion
P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES
Procapsid
Virion
Between
capsomeres
Between asymmetric
units
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 . . .
THERE IS STILL A PLACE FOR X-RAY
CRYSTALLOGRAPHY!
Pseudo Atomic Modeling
Example (Virus + FAb)
Fragment of Ab
CryoEM
+
X-ray
Combination of both for pseudo atomic resolution
IMAGINE THE INFORMATION WE COULD ACHIEVE BY
COMBINING NEAR-ATOMIC RESOLUTION CRYOEM AND XRAY DATA!