An Algorithm Set To Revolutionize 3-D Protein Structure Discovery

One of the great challenges in molecular biology is to determine the three-dimensional structure of large biomolecules such as proteins. But this is a famously difficult and time-consuming task.

The standard technique is x-ray crystallography, which involves analyzing the x-ray diffraction pattern from a crystal of the molecule under investigation. That works well for molecules that form crystals easily.

But many proteins, perhaps most, do not form crystals easily. And even when they do, they often take on unnatural configurations that do not resemble their natural shape.

So finding another reliable way of determining the 3-D structure of large biomolecules would be a huge breakthrough. Today, Marcus Brubaker and a couple of pals at the University of Toronto in Canada say they have found a way to dramatically improve a 3-D imaging technique that has never quite matched the utility of x-ray crystallography.

The new technique is based on an imaging process called electron cryomicroscopy. This begins with a purified solution of the target molecule that is frozen into a thin film just a single molecule thick.

This film is then photographed using a process known as transmission electron microscopy—it is bombarded with electrons and those that pass through are recorded. Essentially, this produces two-dimensional “shadowgrams” of the molecules in the film. Researchers then pick out each shadowgram and use them to work out the three-dimensional structure of the target molecule.

This process is hard for a number of reasons. First, there is a huge amount of noise in each image so even the two-dimensional shadow is hard to make out. Second, there is no way of knowing the orientation of the molecule when the shadow was taken so determining the 3-D shape is a huge undertaking.

The standard approach to solving this problem is little more than guesswork. Dream up a potential 3-D structure for the molecule and then rotate it to see if it can generate all of the shadowgrams in the dataset. If not, change the structure, test it, and so on.

Obviously, this is a time-consuming process. The current state-of-the-art algorithm running on 300 cores takes two weeks to find the 3-D structure of a single molecule from a dataset of 200,000 images.

Brubaker and co have developed a much faster method that can do the same job in only 24 hours working on a single workstation. The technique relies on two algorithmic innovations.

The first exploits the fact that the images are noisy and so contain vast amounts of redundant information. The team gets around this using an algorithm that removes much of this redundancy, leaving only a subset of the original data. The trick, of course, is to get rid of the useless data while keeping the useful stuff, something they manage using a machine learning approach.

That reduces the amount of data that has to be processed but the main speed up comes from the second innovation, a statistical technique called importance sampling.

The main idea here is that certain pieces of data are more important than others in determining the final 3-D structure. So finding a way to focus on those can dramatically speed up the process.

Brubaker and co have found just such an approach. It turns out that large molecules frozen into thin films almost always end up lying on their side. So the shadowgrams nearly always show the molecules in this pose rather than standing on their head or bottom.

Building this knowledge into the algorithm dramatically increases the speed at which it settles on a potential 3-D structure since it can ignore the possibility that the images show the molecule from above or below.

The resulting improvement is huge. “This leads to speedups of 100,000-fold or more, allowing structures to be determined in a day on a modern workstation,” says Brubaker and co.

The team go on to demonstrate their technique on a set of shadowgrams of two well-known biomolecules. The first dataset consists of more than 46,000 images of a large transmembrane molecule called ATP synthase from the thermus thermophilus bacteria. The second consists of almost 6000 images of bovine mitochondrial ATP synthase.

The team also synthesized a third data set by taking 40,000 random shadowgrams of GroEL-GroES-(ADP)7, a biomolecule with a known structure. They then used their algorithm to work backward to recreate the original structure.

Finally, the team compares its approach to other state-of-the-art models and show that the new algorithm significantly outperforms these standard methods.

That is an impressive result that has the potential to dramatically change the landscape for molecular biologists who have struggled for years to find reliable new methods for determining the structure of large biomolecules.

Electron cryomicroscopy looks set to take on this role. And the technique is likely to get better as the resolution of this form of microscopy improves in the coming years.

Ref: arxiv.org/abs/1504.03573 : Building Proteins in a Day: Efficient 3-D Molecular Reconstruction

The logical consequence is fiddling with the human genome. Tell me I am wrong.

Not really. We can already tell through biochemical means the functions of many of these proteins even though we have only a vague idea what the structure is. We have the technical ability to alter the human genome right now. What keeps us from doing that is the ethics of genetically engineering humans.

Many animals have been genetically engineered, usually because changing a function of a gene helps to illuminate what the unchanged gene does.

While I have personally thought that it might be advantageous to have fluorescent kids (you can't find your kid? Shine UV light and you'll see him immediately!), altering humans in meaningful ways is still probably not very feasible. We still do not understand enough about how the human body works to be able to make targeted changes. For instance, we do not understand the complex genetic profiles of intelligence, so how can we engineer "super" humans?

Being able to elucidate more protein structures faster is a big step forward, but it won't lead to Brave New World.

You are wrong —DNA is transcribed to RNA, which is translated into Protein. Proteins comprise biochemical signals (like hormones) and form structures and substructures.

If DNA is like playing chopsticks on a piano, then protein is jazz —it’s much more complex and the unknowns are much, much greater.

New drugs are generally proteins, and in order to understand their functionality before you (very expensively) create new ones, it’s nice to have a good idea of how they’ll fold and funciton in the body beforehand —you’ll save tons of money, that way.

So this new technique will provide researchers a faster, cheaper, more reliable way of visualizing those proteins. They’ll know earlier if the new protein they’re making is useful or promising in remedying some disease.

Ever wrap Chrismas presents with those curly ribbons you made by using a scissors blade to modify a strait ribbon into a super curly ribbon?

Proteins are like that, sometimes with strait regions, then later curling again. Each segment of the protein (amino acids) has a specific size and charge, and that’s what makes it pull in on itself, or push apart, at different locations.

Very big subject here that can go shooting, spiraling and exploding in a fireworks of discussions.

The answer to your question is yes and no depending on what ring of the circus you are viewing and there are more than a few hundred rings to this circus.

My microbiology is a little dated but the elementary dogma (yes, I chose the word ‘dogma’) of cellular function is:

* DNA —> ‘transcribed’ —> RNA
* RNA —> ‘encodes’ —> Amino acid (AA) string in Golgi Apparatus
* AA String Bead —> ‘fold’ —> protein, enzyme, hormone

Ok, so far you can think of the DNA as the stored code, the RNA as the execution code, the Golgi Apparatus as the ‘factory cavern’, Amino Acids as building blocks of proteins, enzymes and hormones.

The terminology can throw newbies off but many of the same things are called different names. For example,

proteins = enzymes = hormones

because they are all 3D structures of amino acids; they just take different names because of the context.

And then there are different labels attached to the same object depending on where in the production line it is. For example, RNA is called messenger RNA or mRNA for short when it transits from the DNA complex to the Golgi factory and then is becomes tRNA at the Golgi because it was ‘edited’ by RNAi or interference RNA ... Whew! Etc. etc, etc,

But terminology helps communication to be precise.

For example, what’s the difference between a circus clown, a rodeo clown and Jon Stewart?

They are all clowns but they clown around in different settings, and the latter one is especially retarded in politics.

But to get an idea of the importance of knowing 3D protein structure, cures of certain prevalent forms of cancer can be realized by knowing precisely the 3D structure of certain proteins or enzymes or hormones.

For example, DNA is replicated to RNA by RNA Polymerase, what’s that? Well, it’s an enzyme, uh... protein, uh whatever. It’s a 3D structure of amino acids that is uniquely adapted to ‘strip off’ RNA that is bound on certain ‘regions’ of DNA and send it on as mRNA.

A picture here is worth a thousand words.

So why is a 3D Structure of RNA Polymerase important?

Very big question with forty or fifty different circus rings to watch.

Here’s one ring of interest: Some cancers are originated from too much or too little RNA being transcribed, called ‘over-expression’ or ‘under-expression’ (more words and terms). For example, Non-Hodgekins Lymphoma is marked by the over-expression of two genes. What’s a gene? Ok ok ok .... you can see that terminology is a huge part of microbiology and there a gazillion primers on the internet that will bring anyone up to speed on it. But look at the cell as a huge factory complex for making and assembling cars and trucks (proteins) and then shipping them off to the roads and freeways of the region where they are used for myriad purposes.

Knowing the 3D structure allows for small molecules known as drugs or biologics, etc. to attach to the ‘macromolecule’ or the Mother Ship and modify its structure or function. Think of dropping a Shelby Mustang Engine into a Fiat. Uh, .... no, don’t think of that. But you get the idea.

Another key thing to know about proteins = enzymes = hormones is that they may come out of the cell as a bead string of magnets with different positions of their polarity lines. So what happens? THEY FOLD!!! THEY GLOB UP! Little magnets go Click! Click! Click! Bam! Bam! Bam! And the result is a very tightly bound glob of magnetic mess.

Why is a knowledge of the GLOB important?

Because it just so happens that all these globs of amino acid magnets have a ‘pocket’ that is constantly in contact with other structures that may or may not ‘fill its pocket’ and activate it or neutralize it. This is known as protein function and is very important to everything. Proteins are born incomplete and function to become complete.

And this ‘incompleteness to completeness’ brings us to the grand mystery of women.

Uh, ... let’s not go there.

But it’s true, humans reflect a grand version of what goes on in the microbiology. Yep, this is part of God’s creation.

So parents, tell your sons to study microbiology before they get married. It might just extend their lifetime.

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