Document 14974293

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Most
you will
associate EM with
pictures like the one
shown to the left.
Courtesy Dr. Marc Paypaert, Yale Univ CCMI
of
Molecular EM-New Frontiers
Tubulin a/b-heterodimer
from 2D-crystals
Coutesy Nogales&Downing
Tubulin model fitted into helical
reconstruction of a microtuble
Coutesy
Nogales,Downing&Milligan
Jan 13,1968
the birthday of
molecular EM
Incidently, the discovery of how EM-images can be interpreted
occurred at a time that saw the first X-ray crystal structures emerge.
…and back then,
X-ray
crystallographic
structures
looked similar to
what EMstructures look
like today.
…so, how comes that X-ray crystallography took off like a “rocket”
while EM was a “sitting duck”?
EM faced a number of unique challenges that needed to be overcome before it could
become a generally useful tool for analyzing the structure of biological specimen.
First: EM requires a vacuum - an environmental constraint that is incompatible with
unprotected biological material.
Electron beam
specimen
negative
stain
image
Historically, specimen were protected against the
vacuum by negative staining procedures.
These protocols exploit that salts of heavy
metals are relatively insensitive towards
electrons and form a stable “cast” around the
molecules when dried down. Salts such as
uranylacetate or phosphotungstic acid titrated to
neutral pH, vanadates and molybdates have and
are still being used.
The granularity of the stain limits
resolution to about 15Å. Also, no internal
detail is discernible because what is
observed is the stain-exclusion pattern
Finding a way around the use of negative stain was paramount for making any more
progress towards meaningful EM-work on biological samples
A simple example: Liposomes
4,400x
liposomes
negative
stain
Electron beam
image
Negative Stain: image shows
stain exclusion pattern
No internal detail/features are
visible
26,000x
Sample Vitrification
If water is frozen very
rapidly, it turns into an
amorphous, solid state
known as vitreous water. In
this
state,
the
water
molecules are disordered
and form a transparent
“glass-like” solid around
the
biological
sample.
Hence, we directly observe
the biological molecule
and its internal features
when imaging in the frozen
hydrated state.
Piston of
plunger
forceps
EM-grid
liquid ethane
vitrification occurs
at 105 ˚C/sec
liquid nitrogen
Negative Stain
liposomes
negative
stain
Frozen-Hydrated
vs
Electron beam
image
Negative Stain: image shows
stain exclusion pattern
No internal detail/features
water
image
Frozen Hydrated: embedding
medium is “transparent”
Internal detail/features are revealed
The Destructive Power of Electrons
After 0.2 sec
1 sec exposure
“In theory, there is no difference between theory and practice.
In practice, there is”
Frozen hydrated
Negatively stained
Particle picking from images of frozen hydrated samples can be challenging…
Low contrast imposes certain minimum requirements on the
state of the sample
Structure determination of small molecules requires crystalline
samples, known as 2D-crystals
That is, in the case of 2D-crystals we can use a crystallographic
approach towards structure. The advantage of having images is
that there is no “phase problem” in electron crystallography!
Electron Microscopy of Biological Specimen
APPROACHES
Crystalline
Non-Crystalline
2D-crystals
Single layer of molecules ordered in xy
plane
Examples: BR, LHCII, AQP1 and tubulin
H+-ATPase, PSII, rhodopsin, gap
junctions
Easiest approach to get
resolution
Resolution range: 3-20Å
to
helical
Examples: actomyosin complex, decorated
lipid nano-tubes, RNA polymerase
Resolution range: 20-35Å
icosahedral
Examples: hepatitis B core antigen (~7.5Å)
Resolution range: 7-40Å
high
helical-crystals
“2D-crystal wrapped onto a cylinder”
Examples: AChR, Ca2+-ATPase
Not so easy to get to high resolution, but
AChR is currently at ~4-4.5Å
Resolution range: 4-35Å
single-particle
Examples: Complex I, GroEL (7.5Å),
latrotoxin dimer (~240kD), ribosome (8Å,
phasing)
Resolution range: 7-40Å
tomography
Examples: mitochondria,
muscle
Resolution range: >40Å
Common to all except for tomography: Averaging!
insect
flight
Use of noisy images require us to
average many individual particle
images. Lets briefly dwell on
averaging and what it means….
The final structure will only
reveal the features that are
shared by all particles.
The need for averaging also
contains a potential danger. Can
you see what it is?
The presence of unrelated objects,
conformational heterogeneity, or
particles with slightly different
compositions (e.g of subunits) all
can invalidate the final result
and/or complicate the analysis
Source: “Biophysical Electron Microscopy” (Academic Press)
Chapter 8 p:300
DIMENSIONS
TISSUES
CELLS
MOLECULES
cm-mm
mm
nm
10-2 - 10-3
10-6
10-9
ATOMS
> nm [Å]
10-10
Light-Microscopy
NMR, X-ray
Electron-Microscopy
Flowchart for a Typical cryoEM-Project
Purified Specimen
Sample Preparation
Thin, Vitrified Specimen
Cryomicroscopy
Structure-Function Relationships
Map Interpretation/Model Building
Fitting of High Resolution Structures
3D Density Map
Image Selection
Digitization
Image Processing & 3D-Reconstruction
Micrographs
Adapted from Baker and Henderson,
International Tables of Crystallography Vol F
Core Facility for Electron CryoMicroscopy
Instrumentation
120keV Tecnai 12 TEM
for screening & electron diffraction
200keV Tecnai F20 Field Emission
TEM
for high resolution imaging
• Accessory equipment necessary
to perform cryo-EM & to allow
high throughput data collection
on both systems
• Optical bench
• High resolution image scanner
• DEC Alpha ES-40 server
and workstations for digital image
processing
• SGI workstations for molecular
graphics (as part of Bioinformatics
Core)
How to get started?
1. Identify specimen:
size (the bigger the better, preferably >500kDa, but can do almost
anything if not afraid of trying to crystallize sample….)
quality: single protein? Yes: >90% pure No: substoichiometric subunits?
stability: the more stable the better. Avoid glycerol if possible….
quantity: negative stain (few µg), cryoEM: comassie stainable (if possible)
2. Get
excited
and
contact
either
Vinzenz
(5-5652,
vinzenz.unger@yale.edu) or Fred (5-5773, fred.sigworth@yale.edu to
arrange for a quick sample evaluation and/or discuss your specific case.
http://cellserv.med.yale.edu/imaging/ccmi/cryo_electron.html
3. Join the user group and have one of your students or
postdocs trained.
4. Publish structure….
How long will it take?
Hard to predict, but can potentially be long-term (1-3 years). May consider
doing a reconstruction in negative stain first (this is a good idea anyway),
which usually takes a lot less time.
How much will it cost?
Training: free of charge, provided you use the facility after training is
completed.
Usage: current fee is set at $30/hr (--> ~$5,000 if you plan to use the EMs
for 3hrs a week). If the project transitions to cryoEM you need to commit
~$10,000 per sample. There are safeguards build into the policies of the
facility that account for unforeseen hardship = in short: don’t worry - come
on in and enjoy!
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