Tag Archives: Personal

Crying over Cryo-EM

OK so the title is hyperbole, but I’ve definitely struggled recently with my cryo-electron microscopy. I guess here I’ll give an overview of what exactly electron microscopy is and why I’ve struggled.

Professor Jensen of CalTech has a great series of videos on Cryo-EM. Why we use it, how we use it, and what it is. Anyone interested in the technology should watch it, but for my own purposes:

  • Cryo-electron microscopy consists of freezing a sample and then shooting electrons at it to see the 3d structure of it at the smallest atomic scales.
  • We’re using it to study a number of proteins that cause diseases. In particular we want to know how the 3d shape of a certain protein creates that protein’s function. And how that function can then go on to cause a disease.
  • So we purify a specific protein, make a cryo-grid from that purified protein, and then look at that cryo-grid under electron microscopy hoping to get a good 3d structure.

But that’s where the problems start. First of all, purifying a protein to 99.9% purity is no small feat, especially when you’re taking proteins out of actual patient samples. I’ve dearly struggled to get the required purity that would be needed to make good grids for imaging.

But once I have some “pure” protein, I need to add it to a grid to image it. A cryo-grid is a 1 millimeter by 1 millimeter circle about 1 micrometer thick. On that grid are cut out many 1 micrometer by 1 micrometer squares. And in each square are a mesh of 100 nanometer by 100 nanometer holes. When I add a tiny drop of my protein sample (which is in water) onto the grid, the hope is that the proteins will settle down into the holes. I will then “blot” the sample by pressing some paper onto both sides of the sample, which wicks away all the water not in the holes. I then instantly plunge the sample into liquid ethane, freezing all the liquid in the holes in an instant.

What you get is supposed to be a grid covered in a tiny thin layer of ice, and in each hole the ice contains your proteins of interest. Since they were flash frozen in ethane, the ice here is “vitreous,” which means glass-like. It’s see-through just like glass. And so a beam of electrons can pass into the ice to create an image of the proteins inside the ice.

But there’s problems. Let’s get back to making the grid: most proteins are hydrophilic which means water-loving. The opposite of hydrophilic is hydrophobic which mean water hating, like oil. Oil and water don’t mix, and neither do hydrophobic and hydrophilic things. Our grids are made of copper covered in a layer of carbon, and that stuff is naturally hydrophobic, meaning it doesn’t interact well with the hydrophilic proteins (and the water they are in).

So before adding proteins we have to glow discharge our grids. This means putting them in a machine that shoots broken-up water molecules at them. Those broken-up water molecules have oxygen in them, and some of them will bind to the grid creating oxygen-containing compounds. Those compounds are very hydrophilic, so the whole grid becomes hydrophilic enough for the proteins to interact with it.

At some point we got a new glow discharger, and I swear that it started destroying my grids. Like I said the grids are tiny and fragile, 1 millimeter across, 1 micrometer thick! This glow discharger shoots water at them, and the new one shot the water so hard that it was punching through my grids and destroying them completely at the microscopic level. I couldn’t see the damage because it’s microscopic, but after adding the protein to my grids and flash-freezing them, I’d look at them under a microscope and see nothing but a completely destroyed grid. I finally just stopped trusting it completely and moved on to using a new glow discharger that’s a bit weaker.

So OK I solved the glow discharge problem, but now here comes the ice problem. Like I said above, you want the proteins to be encased in glass-like vitreous ice. If you have no ice, well you have no proteins. And if the ice is too thick, it’s no longer glass-like and you can’t see through it. I kept being on both sides of those extremes, first I had ice so thick I couldn’t see anything, then I had no ice at all. You are supposed to manage this problem by configuring your blotting time, which is how long you wick away the water before plunging the grid into the liquid ethane. Shorter blot time, thicker ice, longer blot time, thinner ice or no ice at all. Try long and short times to get the ice just right.

And yet I was using ultra-short blot times and still getting thick and thin ice sometimes at random. On the balance I got more grids with no ice at all, so I kept thinking I needed to drop the blot time more and more. My adviser said that there is a minimum blot time of about 2 seconds and you never want to go lower than that, but I tried 2 seconds and the ice was still way to thin or non-existence. That seems to say that my blot time is still too long, yet 2 seconds is as short as I can go.

I finally asked an expert in the chemistry department who suggested I used their facilities instead. He also suggested that 1 second of blot time is perfectly fine, and so that was what I did. I FINALLY seemed to start getting good grids, so let’s hope it hold out.

So I’ve struggled with glow discharging, and then blot times, as well as protein purity. I’ve finally got some good grids, and I hope I can collect a lot of data on them. If I do that, I may be able to get 3d structural information using AI and a whole bunch of analysis. We’ll see though, we’ll see.

Why is it every time I sit down to write, I suddenly can’t write?

I am unfortunately not writing a real post today, rather a post about what I wish I were writing about. Every time I sit down to write anything these days I get bad writer’s block and so I end up not writing or banging out something really mediocre. But I want to write down some of the topics I’d like to write and hope to write about soon as a way to remember them and push me to write about them.

So I’d like to write some posts about the following:

My work using HuggingFace and some other tools to try to fine-tune a large-language model (LLM). I hope one day to get an LLM that can read earnings statements for me and parse out anything interesting. I would go through step by step what I did, and maybe also have a separate post for all the things that really annoyed me (no one would tell me how to make a dataset!)

“Shadow Boxing NIMBYs again” where I talk about why NIMBYism and zoning are bad. This post would in part take in the idea of “people should be allowed to build things on their property,” which is surprisingly controversial. Also tearing about common NIMBY arguments about how corporations and foreigners owning homes are the “real” problem rather than the simple fact that there aren’t enough homes. Also would talk about the insanity of the British “chains” system.

My recent successes and failures in cryo-EM, including the glow discharger destroying my grids and how my ice has been misbehaving. Also the fact that 2 similar samples are acting in completely opposite ways.

This is a bit hard to post

I’m not going to share this post to any of my social media, but I wonder if it would be cathartic to put this out in writing

I’ve been feeling a little jealous of how many of my friends seem to be succeeding in their jobs and their research while I’m not. I’m not getting the data I want so I can publish papers, I’m struggling at writing as much and as well as I would like, and since I don’t work in industry I’m not making as much money or getting the promotional opportunities I want.

I’m just feeling a lot of jealously right now and that’s making it hard for me to sometimes talk about my own trajectory and the trajectory of others.

The danger of small patterns

As I’ve probably said before, I work as a researcher. When you’re doing difficult or expensive research, you don’t usually have the time or money to do a whole lot of replications. That goes doubly if you’re working with patients or patient samples. But since science is all about finding patterns, how can you find patterns in a small dataset?

There are statistical tools that can help with this, but even before you get to the hypothesis testing phase, you need to know which direction your hypothesis will go in. For that, we tend to look at the small patterns which aren’t yet statistically significant and try to see what they mean. The danger here is when you don’t get data in a reasonable amount of time, you want to work on your project but you don’t have data to work on. So you go back to whatever you have, the “small patterns” and start extrapolating from there. “If this pattern holds, what could it mean for this disease?”

Then you can start getting attached to a hypothesis that has no data to back it. When you do get data, you may start to interpret it in light of the small pattern you already detected, a pattern which may not even hold. That’s the problem with small patterns, you get to thinking they mean more than they do.

The human brain is a pattern matching machine. Our first calendars came about from noticing that the seasons of a year came in patterns, and that certain stars in the sky could be seen during the hot season while others could be seen during the colder one. But people also thought they detected patterns about how certain things happened on earth when certain stars were seen in the sky. One pattern between stars and the sky held true, there is a correlation between which stars you can see and the season in your local area. But another pattern was false. Yet both patterns were studied and believed for thousands of years.

I hope I don’t get attached to bad patterns for quite so long as that, but it’s hard to avoid. When you’ve got all the time in the world and not enough data, you get attached to these small patterns that you think you detect. And that can hold true even when the pattern is no longer real.

Invest in what you know? How much do I need to know?

I’m a biochemical scientist. I’ve published papers. I’ve got degrees. As an investor, I’ve often been given the advice (whether from friends or randos on the internet) that to “invest in what you know” is the safest kind of investment. For me personally though, I’ve avoided investing in any particular biotech or med-tech companies outside of passive ETFs, because I feel like while I know a lot about biochemistry in general I don’t know enough in specific to have any kind of advantage in those areas. I know about Alzheimer’s disease, but I don’t know much about pharmacology so how would I discriminate between two Alzheimer’s drug companies I wanted to invest in? I know about CRISPR/Cas, but I don’t know enough about its delivery system in humans to feel confident that I could pick the winners in today’s more crowded CRISPR field. There are a lot of areas of biology that I feel I have a little knowledge, but not enough to give me an edge.

Maybe there’s a Dunning-Kruger effect here though, because while I can’t explain what cloud computing is besides “it’s like renting another person’s computer,” I have thrown a bunch of money into Microsoft and been happily watching it grow. I like my Microsoft products and my office suite, so I feel good enough about them that I feel they’re doing alright. Yet I clearly know a hell of a lot less about Microsoft than I do any of the biotech companies of the world, so why do I feel so confident investing here?

I don’t know, it’s hard to psycho-analyze myself, but am I making all the wrong moves? Should I focus on investing in biotech companies, confident that my background would give me an edge in picking the winners and avoiding the losers? For now, ETFs for me I guess, but I’ll keep blogging about them since they’re fun.

Rest in Peace, Shamus Young

Yesterday, I wrote a post where I offhandedly mentioned the death of Shamus Young.  I had done so because during the post I remembered a decade old post of his (that I still can’t find!) that had so succinctly explained everything I was talking about.  I finished the blog post, and while looking for Shamus’ post so I could link to it, I learned that he had died last year. 

I’m pretty late to the party on this one, I haven’t been following him for a few years.  But I first heard about him when he published DM of the Rings around a decade and a half ago, and for a while he was my main source of gaming news and reviews.  I first played Oblivion because of a post he wrote about it, same with Morrowind and even the original X-Com.  I got a lot of my early gaming exposure from him and his blog, and I still think a lot about some of the things I read from him.  My previous post on Skyrim is based heavily on a post I remember him writing about Oblivion and RPGs in general, and a lot of the concepts he wrote about still come back to me.  Learning that he had passed, at what seems like an early age, kind of hits me.  I was never more than a lurker to his blog, and I don’t really know what I wanted to say with this post.  I’m so late to the party and was no more than a reader, so I can’t really share in the grief with others.  But I just wanted to say that he was incredibly fun and funny, and I’m glad I got to read him.

If you’ve never read it, DM of the Rings is well worth your time.  Farewell to a really cool guy.

Writer for sale

Very small post today because I forgot to write one, but I hope tomorrow to write about Beam Therapeutics so watch this space!

Anyway, I have a job, but I’m always open to new ones. I’ve looked at becoming a stock writer. I know it’s not glamorous or 6-figured, but writing about biotech and pharmaceuticals is a something I enjoy and a job I think I’d be good at. Don’t take this blog as the only signal of my quality (or lack thereof) I write most of these last minute because I have my own 9-5 right now. But I think some of these posts that I’ve spent time on are actually good, and so if any of my readers know of good places that would pay for freelance writers, hit me up at theusernamewhichismine@gmail.com.

Buses have only gotten worse during my adulthood

I’ve lived in cities my whole life and yet it seems like the bus systems in every city I’ve ever lived in have only gone downhill. When I first went to grad school, I used buses and trains to get around town to do my grocery shopping and whatnot. By the time I was graduating, the buses had all gotten so sketchy that I no longer used them. People smoked openly on them, there were always homeless people panhandling, and they just seemed a little too violent to be safe. I eventually left that city for my current one, and the buses are STILL crap but in another direction. They’re never on time, I’ve had multiple days where a bus just plain doesn’t show up, and now they’ve altered the route schedule to ensure that my bus MUST take a much longer time to reach it’s destination. When I started riding my route, I could get from A to B in 20 minutes give or take. They’ve now altered the route to make A to B take 30 minutes, and if traffic isn’t bad and the bus is a little early, the driver will stop on the side of the road to ensure it takes no less than 30 minutes.

I don’t know what buses have always been this bad, but it’s really putting me off public transportation in general. It’s even more galling when the cities I’ve lived in are demanding ever increasing funding for ever worsening service. At what point should the city cut its loses and say no, no new funding without fixing what you currently have. More money isn’t a cure all, countries other than mine have much better bus service at much lower cost, I know, I’ve been to them. The cities I’ve been to seem to have a cost disease, where they keep spending more and more to get worse and worse and the only conceivable cure is more money. It’s infuriating.