u/AAAAdragon

I have been finding missing cats after 6 pm when animal control says they stop working. Why?

I am wondering if there are some local vet clinics open 24 hours or at least till 9 pm which will accept found cats? So that the cats can be taken to the humane society the next day to have their microchip read to notify their owners?

There are enzymes like DNA/RNA (Polymerases, Primases) and nucleoside triphosphate pyrophosphatases that release pyrophosphate. The product pyrophosphate is detectable when the coupling enzyme pyrophosphatase converts pyrophosphate into two phosphates which can be colormetrically detected with malachite green.

A nucleoside triphosphate pyrophosphatase catalyzes the metal dependent hydrolysis of certain NTPs to the products NMPs, pyrophosphate a proton.

One could accurately measure the nucleoside triphosphate pyrophosphatase enzyme’s kinetic parameters K_M and k_cat by measuring the amount of product phosphate in a coupled malachite green assay with pyrophosphatase.

One could also measure the 50% inhibitory concentration of the target enzyme with the coupled assay and an inhibitor of interest. But maybe the inhibition is actually because the inhibitor is specifically inhibiting the coupling enzyme pyrophosphatase’s ability to hydrolyze pyrophosphate into phosphates?

Have people ever published that the target enzyme was inhibited when the coupling enzyme actually was inhibited?

This is keeping me awake at night.

Thanks!

This is not the first lab I have worked in but is the first one which uses Shared Network Drives and this is a really a game changer.

Our lab members process experimental data, analyze the data and put the processed and finished work on shared network drives. We have separate shared network drives for each major project and folders for the subprojects. Each of us has a subfolder for each project for the unique work that each of us do.

If a new member joins the lab, we simply connect the new computer to the shared network drives and that new lab member has read access to all the work of our lab. If a member leaves our the lab, we don't have to email the staff member who left the lab and actually beg for their information because we have read access to all their processed data. The Shared Network drive is basically directories so it works in the file explorer or command prompt and works on any operating system Windows, MacOSx, Linux.

Shared Network Drives really help because our supervisor is caught up to date on what we are working on and by being able to see your coworkers processed and finished work then we don't redundantly do experiments and we don't have to ask them what they did because we know.

Our system prevents us from not knowing what or how former lab member mysteriously did work 8 years ago. So we don't lose internal knowledge when somebody leaves.

I am especially interested if you reunited with a friend you have history with and then you lived happily ever after.

Thank you.

So, a little over half a year ago, I made a post in this group showing you an example of what amazing electron density looks like for a ligand bound to a protein at 1.17 angstrom resolution: Example of what amazing x-ray electron density looks like for ligand bound to protein with high confidence : r/labrats

There is this myth spread among structural biology labs that you need high resolution crystal to see your ligand bound. If you are looking at any 2.85 angstrom resolution structure, if you see a blob with non-continuous electron density and no well-defined shape, know that no, that is not your ligand. If your ligand was bound, you would be able to see that clearly even at that resolution. This myth needs to stop!

While yes, it is indeed easier to make out where the protein backbone, side chains, water molecules, metals, molecules from solvent, and ligands are bound in high resolution data about 1.0 to 2.4 angstroms, if ligand is bound you can obviously see it no problem at lower resolution like 2.85 angstroms as shown in this example. Crystallographers usually aren't comfortable depositing a structure at resolution worse than 3.0 angstroms because at that resolution, you can barely see most of the side chains.

But for a ligand like UDP which has a pyrimidine ring if you can see density of phenylalanine and tyrosine then you can see density for this ligand if it is bound as it is in this example with PDB code 4q12 (RCSB PDB - 4Q12: Crystal structure of a putative uncharacterized protein Rv3404c and likely sugar N-formyltransferase from Mycobacterium tuberculosis bound to uridine diphosphate). If you are looking at a ligand structure quality assessment for this example, this UDP molecule is definitely bound as it has a 98% goodness of fit quality! There is no publication for this structure as high-throughput crystallography groups quietly deposit structures to the PDB. The experiment tab tells you the protein crystallized at 19 mg/mL with 2 mM UDP from CHST screen condition E1, 2.0 M NaCl, 10% PEG 6000 with 20% ethylene glycol as cryo-protectant. Yeah, it would be nice to have a publication with the entry but the raw diffraction data like the actual x-ray spots was deposited here: Diffraction project datasets 4q12.

Here is an image of the structure 4q12 with a green/red polder omit map contoured to 5 sigma and the 2fo-fc map contoured to 1 sigma. Read the captions of the image for the fine details. I calculated the polder omit map which excludes bulk solvent around the omitted region which in this case is residue UDP. I needed the PDB file and mtz file to run polder within phenix. I obtained the mtz file from PDB REDO for this entry 4q12 (PDB-REDO for PDB ID 4q12).

How to generate a polder omit map in Phenix: Generating a polder map

Example of Polder omit map for x-ray protein crystal structure with PDB ID 4q12. The green/red map which is contoured to 5 sigma is the polder omit map which is an omit map that excludes bulk solvent around the excluded region residue UDP. The blue map is the 2fo-fc map contoured to 1 sigma. The map radius is 12 angstroms to show the region around the residue.

It makes sense that UDP is bound here because a phenylalanine ring is stacking against the uracil base and a histidine ring is stacking against the ribose sugar of the UDP molecule. Plus the electron density is obvious. This molecule is not dTDP because there is no density for a methyl projection on the DNA base. It could conceivably be CDP because the molecule has a similar shape that would fill the election density but we know the protein was crystallized with 2 mM UDP. It conceivable that this protein could bind CDP, but we can clearly see that ADP or GDP does not fit the electron density because a purine ring is much larger than a pyrimidine ring and there is no green polder omit map electron density for a purine ring.

So, no obtaining a better diffracting crystal to a higher resolution from the same crystallization conditions won't show your ligand bound if you don't see it clearly bound in a 2.8 angstrom resolution crystal. This myth needs to stop!

Try co-crystallizing your ligand at a higher concentration or soaking the ligand into apo crystals, or try a crystal formed from different crystallization conditions because for instance sulfate or phosphate from ammonium sulfate crystallant can bind instead of the phosphoryl groups of nucleotides. If sulfate or phosphate was bound, you would see it clearly bound. For proteins which bind two ligands, unless you have kinetic data that shows that enzymatic mechanism is not sequential it could be that your ligand of interest will only bind when the other ligand binds.

Visualization software is Coot (Crystallographic Object-Oriented Toolkit).