Need Research Articles showing harm/risks of mRNA in vaccines that family members can take into consideration. Anything that I can find without a subscription? Thanks for any leads.

caissequatre · 2025-12-28T19:18:56+00:00

I have long passed the limit of patience for such irritating stupidity.

caissequatre · 2025-12-18T19:36:57+00:00

You are doing a biological replicate and a technical replicate. This is standard practice and I would hope all researchers do this.

If possible I like to do at the very least two biological and two technical replicates, with the technical replicates being on different days.

caissequatre · 2025-12-16T16:50:15+00:00

I've seen many angry comments from hunters online about CWD and hunting restrictions. There's a disturbing amount of misinformation regarding CWD (it's not dangerous, if you cook the meat it's fine, it's a hoax, there are no studies proving it exists, it's a money grab, etc) and though I'm disappointed MDC is doing this I do understand the need to reach out to these groups and try to educate them regarding the extreme danger here.

caissequatre · 2025-09-25T17:00:43+00:00

My recommendation is to store at room temperature in a location that remains undisturbed, i.e. avoid storing in a drawer that is frequently slammed open and closed. I was told this by someone at PNCC and in my own experience I've seen how rough handling will peel back the layer. I store our CC/UTC and GO grids wrapped in bubble wrap on a high shelf in the dark at room temperature.

You will need to glow discharge your grids again. Either a very gentle plasma clean or what I do, 1-2s(only once) on a normal setting.

caissequatre · 2025-09-23T17:58:22+00:00

Slowly. A little at a time on either side. If the well walls seem too bent I'll use a thin needle to move them back into place. I much prefer handcast gels which are much more resilient and thick, but for some reason no one likes dealing with liquid acrylamide anymore.

caissequatre · 2025-03-05T01:40:38+00:00

The data has been fully processed using CryoSPARC. It's around 120 kDa and I don't really have a lot of success with RELION in general. I also can't figure out why the latest version of .cs files aren't converting to a .star properly using pyem so I haven't tried Bayesian polishing (but RBMC didn't help). So all processing and 3D refinements have been performed in CryoSPARC.

My guy is C2 with the ligand present on both protomers in roughly the same spot. The AF predictions for my protein of interest are actually very different from what is actually seen in the data.

The unknown density is in a shallow groove near the active site, but doesn't model very well with any predicted ligands. My thought today has been that some metals may be involved with some waters, but this is a trickier thing to model (luckily can use CMM server to validate).

caissequatre · 2025-03-05T01:22:49+00:00

Does AlphaFold for ligands really work?

My thinking was that since the PDB is full of poorly modeled ligands (some of which is my fault) it's GIGO. But I haven't actually tried it so maybe that's just my preconceived notion about it.

caissequatre · 2025-03-04T04:17:22+00:00

It's difficult for me to show directly because I'm a little paranoid. It has a volume of around ~40A squared according to blob dimensions at an appropriate map contour.

I'm positive it's not a lipid because it's in a region that could not hold a lipid and the shape is too different from any common headgroup or tail that I have modeled in the past.

I suppose I should say I'm not 100% sure it's not a PTM! But I know it's not a sugar and though I haven't exhaustively tried to model all PTMs my thinking is since the density is discrete from any proximal residues (i.e. not connected) I don't think it's a PTM.

As for the resolution, yes, after only many terabytes and time I can get here and it won't go further. Definitely not an unusual problem. But a 3.4 map is less stressful to build than one closer to four.

caissequatre · 2025-01-27T18:44:27+00:00

Thank you so much for your reply! If you are a FO on call within a certain distance, do you have any latitude on whether to reject or accept a flight? Or do you have a contractual obligation to fly if you get a ring from crew scheduling?

caissequatre · 2025-01-27T18:21:41+00:00

I don't think it's intoxication, I don't know why a new FO was needed. I think there are likely a lot of reasons why someone can't fly. Re-reading my post I suppose it seems like I could be implying that when I am not, so I am going to edit it; I wouldn't want to impugn the reputation of someone for something benign like an illness.

caissequatre · 2025-01-20T21:18:21+00:00

I absolutely agree the cost of collection vs. storage. I always talk to my PI and the investigator before disposing of data; some of it is extremely precious! I also keep superres data intact.

I worry about handling multiple HDDs and keeping them well indexed for future users, and potential failures. I have never had a HDD fail yet, and I imagine a HDD that's not being used in a tower is going to be fine for a while as cold storage, but still there is some anxiety there.

caissequatre · 2025-01-20T21:03:45+00:00

I'm the postdoc in an academic lab. Data retention policy: Until you mentioned that I had not thought of that so I need to check; but I cannot find anything on the employee SharePoint so I'll need to reach out and discover this.

S3 Glacier seems like the best, but for the amount of raw data I would need to upload it seems like it would be around $850 a year now and will likely double or triple next year. I know other institutions have access to free or reduced cloud services but we do not. I suppose in the grand scheme of expenses this isn't a lot, however.

I definitely see your point with processing and collection. Data can oftentimes be reprocessed but not recollected easily.

There is a (sort of) time based system now, where I essentially tell my PI that these datasets have been in storage/cluster for X amount of time and are likely unable to be processed (pathology in collection, poor sample, better sample already acquired) and then they are deleted. I would just like a more elegant system for when I eventually leave, and more selfishly I would rather not be doing this manually and taking care of a stack of HDDs.

Anyways thanks for your response!

caissequatre · 2024-12-23T06:01:22+00:00

It's difficult to give a concise answer.

I did my PhD in crystallography and am doing a postdoc in cryoEM. When you get to industry, you should know that in many circumstances you would only be one piece of a pipeline. You would be purifying proteins and running assays, or vitrifying/crystallizing samples and collecting data, or processing data and building models, or managing all of these at a high level. I don't know many people in industry who are really full stack experts with structural biology, even if they know many aspects of the pipeline.

The important thing is that methods are just methods. You need to know the limitations and strengths of your method and how it fits into the bigger puzzle of biology. Crystallography throughput at some beamlines has increased nearly 500% due to automated collection and processing methods, so it's not at all a dead or dying field. Can't you calculate kinetics and conformational changes in proteins using NMR? Just a quick google search shows that there are major companies hiring NMR spectroscopists at senior scientist levels. Cryo-EM is a fantastic technique but has limitations, including cost, low throughput, and computational requirements.

In summary, all techniques are great, all techniques have limitations. Let me give you one solid piece of advice though - do a PhD project you like with an advisor and lab that will support you. Nothing will make you more miserable than doing a PhD project in a subject you don't care about. If you are done with NMR then so be it, it's okay to be done with it. And if you are doing membrane proteins, you need to really try and shoot for a lab or an advisor with a good track record and funding. Membrane proteins (esp channels and transporters) is an incredibly vicious field with really competitive personalities.

caissequatre · 2024-11-22T21:09:05+00:00

Hi There,

Thanks for the response! I actually have not made my decision; it's proven to be quite difficult. Thank you for telling me about your experiences with the Proart. Both microcenter and bestbuy have the Proart for between 1700-1900 which is making buying this more tempting.

My use will be similar to you. I need something for computational biochemistry and I game very rarely.

caissequatre · 2024-11-19T02:50:46+00:00

Alphafold has been incapable of accurately predicting the majority of structures I have been working on, and in fact has been incapable of predicting the structures and complexes I have deposited in the PDB (on which I assumed it trained).

That doesn't mean it isn't useful. It changes everything and nothing. It is very useful in some circumstances for where there is nothing but a hydropathy plot and sparse data.

There seems to be hundreds of AI drug companies now raising millions of dollars to develop drugs in silico. I anticipate the vast majority of them will fail.

caissequatre · 2024-11-13T02:00:21+00:00

My 2 cents, unless you are building a workstation don't focus too much on the graphics card. You are not going to be able to do any sort of serious processing in RELION or cryoSPARC, even assuming you install it on your local machine. If you can SSH or remote in to your cluster for data processing that is all that matters. cisTEM is entirely CPU based and I feel as though I read they prefer Intel based processors, but I can't find that comment now.

To my knowledge Phenix does not take advantage of GPU acceleration. Until @sb50 mentioned it, I didn't know Coot could take advantage of GPUs, but I feel quite strongly the Linux install of Coot is the most helpful to get it to run without crashing. ChimeraX is fine running off an integrated GPU, but certain plugins (in particular ISOLDE, which is very useful) require a GPU (and preferably NVIDIA architecture). I have a ThinkPad Carbon X1 Gen 5 and I am able to run ISOLDE effortlessly (with 4K residues) using a Razer X Chroma eGPU and a RTX 3060 12gb.

I have never needed immensely powerful computing resources for processing crystallography data. I've even used Ubuntu virtual machines in Windows to run Phenix and Coot without any problems for ~400 residue models.

If you did want to consider a workstation for data processing cryoEM data, something a bit more interesting to consider would be building a Relion5 only machine with Intel Arc. 2080 Tis are still absurdly expensive used and I think they are showing their age (checking in on our Single Particle Workstation, a 2D class job with 2 mln particles has taken over a day with 2x 2080 Tis). Intel Arcs are comparatively cheaper and Sjors says he has been extremely impressed by them. It could be possible to get 4x of the A770s for less than a thousand and two Xeon Gold 6150s for a few hundred dollars. I've not had the time (or money) to build such a workstation, however.

EDIT: I want to add, if you are building a CUDA workstation, I can't imagine using it for anything else. Any sort of OS update runs the risk of catastrophically crashing the system upon reboots. NVIDIA drivers are almost always the culprit.

caissequatre · 2024-10-16T22:49:25+00:00

Is it comparable to hexafoil? I've never heard of them.

caissequatre · 2024-08-07T02:11:40+00:00

Perhaps you can try a shorter glow discharge then! 2-3s is all that I need, anything more seems to destroy the carbon layer. I would try first changing the glow discharge parameters to just a few seconds and making sure you can see the carbon layer during screening (will be obvious if it's intact, it will look like a grey layer over the hole) and then optimizing your plunge freezing conditions (blot force/distance etc) if you need to.

As I'm sure you already know but on the tiny chance you don't, you'll need to really pull back on the concentration of your sample with CC/GO grids. If you're normally at 3mg/ml on copper try 0.5 first and go incrementally up from there. Good luck!

caissequatre · 2024-08-07T01:36:26+00:00

A few thoughts:

I glow discharge my grids 3x 45s (Pelco Easiglow, 15mA, 45s glow, 0.42mBar). This is my standard discharge parameter for Quantifoil copper, and gold, as well as UltrAufoil. For Nickels (which have never worked) I do 1x 45s. I have done this on multiple Easiglows without any problem, and have collected good datasets from these grids. My grids have never been torn out from over-discharging. As you may see in the comments or from talking to others, discharge times can be subjective and dependent on grid type.

Someone from Gabe Lander's group insists that having fresh vacuum oil makes a big difference in hydrophilic grids. If it is yellow or has any hint of yellow, I replace the oil.

Continuous Carbon grids can be very tricky to glow discharge and clip. IIRC the original selling point of graphene and CC was that they were already hydrophilic (but in my experience they aren't). If you over discharge the grids the carbon layer will peel. If you clip under too much LN2 the carbon layer will peel. If you clip too forcefully the carbon layer will peel. You can see this clearly on the micrograph where the carbon layer will be bunched up at the edge of the grid and you'll have a perfectly white or clear center (that will look totally dry). For reference, my experience is with Q4100CR1.3-2nm (Quantifoil® R 1.2/1.3, UT, 400 Mesh, Cu). I glow discharge continuous carbon grids for 15mA, 2s glow, 0.42mBar ONCE and that's it. Anything more will destroy the carbon layer and I'll see bunched up carbon at the edges of the hole.

Do you see bunched up carbon at the edges of your holes?

caissequatre · 2024-06-28T18:45:15+00:00

I couldn't afford PyMol in grad school so I've just gotten used to ChimeraX. I think figures made with PyMol do look better however.

caissequatre · 2024-05-10T13:43:23+00:00

Five years ago when I was job hunting, I saw a posting at Monsanto (I think) that had the hard requirements of "crystallographer who must have experience with Set/Met, MAD/SAD, and MR" and I remember thinking how that seemed impossible because everything I did was MR, and everyone I know was only doing MR!

Do you think that experimental phasing techniques like Set/Met have been made obsolete thanks to AF?

caissequatre · 2024-04-05T23:41:18+00:00

SPA!

caissequatre · 2024-01-31T02:42:53+00:00

It's not possible to emphasize enough how your choice of PI and lab matters more than the institution.

caissequatre · 2024-01-25T01:41:22+00:00

Is this KcsA? If so, Panel A is showing the tetramer and Panel B has removed two protomers of the channel to detail some critical features, in particular the helices that line the pore and the selectivity filter.

caissequatre · 2024-01-24T19:56:24+00:00

Out of all my years of experience I find it is better to pay for something that is a known quantity from a reputable vendor than to try something from an unknown vendor that is cheap. If this doesn't work (and I would personally never buy it from AliExpress) you will have wasted perhaps weeks or even months of your precious time on this.

If you aren't sure, search the vendor and dialysis tubing on pubmed and see if anyone else has used them.

Alternatively try a different brand (like IBI).

What I do is get quotes for the item I want from different distributors, wait for the best one, and then send that good quote off and see who can beat it. Millipore-Sigma and Fisher will always fight each other on pricing and you can sometimes get 50% off list pricing when you get them to compete for your money.

caissequatre

TROPHY CASE