This class covers the fundamental principles underlying cryo-electron microscopy (cryo-EM) starting with the basic anatomy of electron microscopes, an introduction to Fourier transforms, and the principles of image formation. Building upon that foundation, the class then covers the sample preparation issues, data collection strategies, and basic image processing workflows for all 3 basic modalities of modern cryo-EM: tomography, single particle analysis, and 2-D crystallography. Philosophy: The course emphasizes concepts rather than mathematical details, taught through numerous drawings and example images. It is meant for anyone interested in the burgeoning fields of cryo-EM and 3-D EM, including cell biologists or molecular biologists without extensive training in mathematics or imaging physics and practicing electron microscopists who want to broaden their understanding of the field. The class is perfect as a primer for anyone who is about to be trained as a cryo-electron microscopist, or for anyone who needs an introduction to the field to be able to understand the literature or the talks and conversations they will hear at cryo-EM meetings. Pre-requisites: The recommended prerequisites are college-freshman-level math, physics, and biochemistry. Pace: There are 14.5 hours of lecture videos total separated into 40 individual “modules” lasting on average 20 minutes each. Each module has at the end a list of “concept check” questions you can use to test your knowledge of what was presented. As the modules are grouped into seven major subjects, one reasonable plan would be to go through one major subject each day. That would mean watching a couple hours of lecture and spending another hour or so thinking through the concept check questions each day for a week. Another reasonable plan would be to go through one module each day for a little over a month, or even three modules a week (Monday, Wednesday, and Friday) for a 3-month term. It is likely that as you then move on to actually begin using a cryo-EM or otherwise engage in the field, you will want to repeat certain modules.
Introduction: 2-D Crystallography and Sample Prep
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4.9(224개의 평가)
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SS
Jan 26, 2017
I have worked a bit in cryo-em before and I find this course absolutely amazing! Very detailed and easy to understand.
BA
May 16, 2016
Very well detailed on a easy to learn pase. Will help you learn all to EM that is necessary before using them.
수업에서
2-D Crystallography
강사:
Grant J. Jensen
Professor of Biophysics and Biology; Investigator, Howard Hughes Medical Institute
Now that we've discussed tomography and single particle analysis.
The third basic modality in 3D EM is electron crystallography or
sometimes, 2D crystallography and two-dimensional crystallography
is the preferred approach when you can purify a large number of
copies of your object of interest and you coerce it to crystallize.
And the first thing we'll talk about is how do you get a protein to crystallize
into a two-dimensional crystal.
So part of the answer is that some proteins naturally assemble into
2D arrays that are immediately useful for 2D crystallography.
So, an example of this is the protein bacteriorhodopsin.
Bacteriorhodopsin is a integral membrane protein with seven
transmembrane helices and certain bacteria express so
much bacteriorhodopsin that they pack their membranes full of it and
it spontaneously forms a well ordered 2D crystal.
As can be seen in this image from Niko Grigorieff where one of these crystals was
imaged and so you can see this is a bacteriorhodopsin molecule.
Here's another one and here's another one.
So they form trimers and then these trimers organize themselves.
They're closely packed into a beautiful 2D crystal.
Another example of a protein that naturally assembles
into a well ordered thin 2D array is aquoporin.
Aquoporin is found in many places.
In one case, it's present in the membranes of the lens of your eye.
And there it's found to bridge between two membranes.
So here's one membrane and then there's a gap and there's another membrane.
And aquoporin is found to bind across these membranes.
Here's one aquoporin molecule binding to one in the other membrane and
link them close together.
And in addition to linking close together across the membranes,
they also self-assemble in to well ordered arrays within each membrane.
And so looking at them in the electron microscope, this is an aquoporin crystal.
It's very flat and large and the proteins are already well ordered.
If however, you want to crystallize a protein that doesn't naturally
self-assemble into well ordered 2D arrays, one approach that can
be taken has been called the Lipid Layer Crystallization technique.
And here, you have a solution of your protein and
you deposit a monolayer of lipds on the interface betweenthe water and the air.
The hydrophobic tails stick up towards the air and
the hydrophilic heads embed into the solution and form this nice monolayer.
Then if your protein of interest has some affinity for the hydrophilic head groups,
the protein will concentrate right on the air water interface.
And because the lipid monolayer is still fluid, the proteins can still rearrange.
And so, if there's by chance a nice contact that causes one to
bind in a regular pattern up to its neighbor,
this can propagate and into forming a 2D crystal.
So here, for example is an image of a 2D crystal of RNA polymerase two,
which was formed by this Lipid Layer Crystallization technique.
One of the ways to engineer a nice interface between the lipids and
the protein of interest is to use nickel NTA lipids and
then a his tag to your protein of interest.
And once the crystal forms on the air, water interface, then sections of
that crystal can be picked up on EM grids, represented here by this slab.
You can carefully pick up those crystals onto the EM grid.
Now, once the 2D crystals are on the EM grid, they can be stabilized by being
embedded in either some material that can dry and remain at room temperature or
they can be embedded in vitreous ice through plunge freezing.
So here's an example for instance, from this paper in Methods
in Entomology, where several 2D crystal seen here.
Here's another 2D crystal, here's a third 2D crystal have been
embedded between two layers of continuous carbon film in a trehalose solution.
So you might have a continuous layer of carbon and then a solution with your 2D
crystals in it and into that solution, you can add a sugar like trehalose.
And then as that dries, you can add another carbon film on top of that and
let the water dry and it embeds the 2D crystal in the sugar sandwiched
between two layers of carbon and that's one way that they can be observed.
Alternatively, the 2D crystals can be spread into a thin film on the EM grid and
plunge-frozen, just like we've described in other circumstances.
So here is the extent of a 2D crystal that has
been plunge-frozen on a quantifoil grid.
You can see the regular pattern of holes in that carbon surface.
This is just a beam stop that's been inserted into the field of view, but
this is what a frozen hydrated 2D crystal looks like.
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