Re: [ccp4bb] reindexing mtz

Just to be absolutely clear about my approach to doing this, I should scale in HKL2000 as an unmerged set, and then take the unmerged scalepack, convert to mtz and run through pointless? Also, would taking my current mtz (which is a merged dataset) work using the reindex program in CCP4? Are t

[ccp4bb] reindexing mtz

Dear all, Looking to get some advice on reindexing an old dataset. I recently solved a structure of reasonable resolution at ~3A in C2. I had an old dataset at 4A in P213 that I never got a MR solution with until I solved the structure of it at 3A. I've recently gone back to to this and managed

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

Hi Sutapa, With regards to insect cell expression, how do you know it wasn't expressed? You didn't see a band by coomassie staining or by western? If by staining, I'd recommend to just try expressing a limited amount through infection of a few 150mm dishes of your favorite cell line (I use Hig

[ccp4bb] off topic: MacPymol and macOS Sierra

Hi all, Sorry for the very off topic message, but I recently upgraded my OS to Sierra and all of a sudden MacPymol has stopped working for me. Clicking on the program just gives the appearance of it about to start by showing the icon on the dock, and then just blinks out, without ever starting

[ccp4bb] off topic: good peak on gel filtration

Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughl

Re: [ccp4bb] offtopic: steady state kinetics

Over the last 12 hours I've received a large number of responses, and I'd like to thank you all for the detailed and helpful responses. Clearly, I've been doing this incorrectly, however, I do have a number of experiments already done that hopefully I can salvage something out of. Now, at high

[ccp4bb] offtopic: steady state kinetics

Hi all, Sorry for the incredibly dumb and off topic question. I've been doing some steady state michaelis menten kinetics, and no one around me has ever done it before. My own classroom training was years ago and so I've forgotten a great deal and have just been going from old textbooks/papers.

[ccp4bb] Offtopic: Trp as a nucleophile?

Hi all, This is probably a really bad question, but are there any examples of Trp acting as a nucleophile at all in literature? I can't imagine it can, but I thought I'd ask just to make sure from people who know more than I. Thanks and sorry for the offtopic question. Peter

[ccp4bb] oof topic: pH effect on substrate analog

Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing >90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) w

[ccp4bb] off topic: reproducing hits from organic solvents

Hi all, I recently got some hits from a Mosquito grown at 18C with PEG4000 and 10% propanol. I've tried reproducing these hits using the standard 24 well format with no success, even with playing with PEG and propanol concentrations. The initial screening solution was from a kit that's been sit

Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?

tability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, "Peter Hsu" wrote: Hi all, I've been purifying my protein off a GST column and hav

[ccp4bb] off topic: reduced glutathione interfering with protein activity?

Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glut

Re: [ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

I too am also studying the reaction mechanism of an enzyme, but my chemistry/enzymatic biochemistry is rather weak after many years of non-use and no review. Does anyone know just as a general rule which residues are the best to worst nucleophiles? Sorry if this seems rather presumptuous, just

[ccp4bb] offtopic: packing gel filtration columns

Hi all, Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and

[ccp4bb] offtopic: AKTA prime

Hi all, We've got an old Akta prime that I think is on the verge of kicking it. Hearing some high pitched sounds coming from the pump when we're running it. Line A seems clogged and makes a thudding noise when we try to do a pump wash through that line. Does anyone have any experience w/fixing

Re: [ccp4bb] help regarding structure solution

Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see w

[ccp4bb] nonspecific interactions with GST beads

Hi all, I've recently switched over to using a GST system for purifying my proteins. Although I can get enough protein out of a few liters, I've always been finding that when I take some of the beads to run on a gel, I always get a decent amount of protein just stuck on there that won't elute o

[ccp4bb] metal modelling in coot

Hi all, Forgive the rather ignorant question, but I'm rather new to using coot/model building and I'm trying to model in a Zn2+ atom but coot doesn't seem to have the files for a Zn atom in its database. I'm not sure where to start looking/how to write a file that defines a Zn2+ atom and to hav

[ccp4bb] commercially available proteins

Hi all, Sorry for the very off topic problem. I'm looking for a loading control for my westerns (doing in vitro assays). Due to separation/antibody specificity issues, I need super good separation between 70 and 100 kDa, oftentimes running those markers all the way to the bottom of the gel. If

Re: [ccp4bb] zinc fingre

Hi Rajesh, Have you looked at how well conserved these Cys/His residues are? Is the spacing similar to known zinc fingers? Might be good things to consider if you suspect a zinc finger in your protein, of course you probably know this already. Best, Peter

Re: [ccp4bb] effects of salt on twinned crystals

Forgot to mention, that this 2.5-3A diffracting crystal was the same one that I have been unable to index and suspect are twinned due to the presence of these other fused crystals in the same drop. Thanks for any input.

[ccp4bb] effects of salt on twinned crystals

Hi all, Thanks to all those who responded to my query about indexing my dataset. I'm beginning to suspect that the crystals are twinned. the crystals initially started as fused plates which were then optimized to single crystals that diffracted poorly (3.5-4). I then set up an additive screen,

[ccp4bb] HKL2000 indexing problem

Hi all, I recently collected a data set off a single crystal and have had problems with indexing it. Every time I go pick peaks for indexing it constantly picks peaks that are just slightly off the actual peak. After indexing, it would always be that 2 of the 3 cell dimensions would be fairly n

Re: [ccp4bb] His Purification

Have you tried dialyzing the "purified" protein after elution into a no imidazole buffer while doing TEV cleavage, and then reloading on to a His column to remove the tag and TEV? I've found in my experience that this often recaptures a lot of the background proteins. If you're getting a 70kDa

[ccp4bb] Off topic: Beryllium chloride

Sorry for the very off topic and dumb question, but does anyone know if BeCl2 needs to be prepared fresh for use (making BeF3) or can it be stored as a solution stock at room temperature/frozen? Thanks, Peter

Re: [ccp4bb] Complex seeding

I forgot to mention, I can reconstitute the complex (co-expression/mixing proteins) and the complex comes off both ion exchange and an SD200 as one peak. Just haven't had luck with getting crystals.

[ccp4bb] Complex seeding

Hi all, I've been trying to crystallize a 3 protein complex recently with little success. However, crystals of each subunit have previously been crystallized. I was wondering if any one knows of any literature/experiences where people have used seeds from an individual subunit to seed for a com

[ccp4bb] Natrix screen

Wanted to get an opinion. I set up a screen a while ago and got these very small cubic looking crystals 2-3 days later amidst a lot of precipitant. The condition was Natrix E1 (0.04M LiCl, 0.02M MgCl2, 0.04M Sodium Cacodylate pH5.5, 30% MPD, 0.02M Hexammine Cobalt (III) chloride). This protein's

Re: [ccp4bb] Question about TEV cleavage

We've been using the S219V mutant for cleaving out tags. We usually do our cleavage reactions in an overnight dialysis after a Ni-column into 50mM Tris, 100-200 mM NaCl, 5 mM BME at 4C. I've never found problems with losing any protein in the reaction and recover usually >90% of the protein afte

Re: [ccp4bb] off-topic: tag removal

I haven't used it personally, but I've heard some people engineering a his SUMO tag on to the protein and then use SUMO protease to cleave it off. The protease apparently makes very clean cuts and no extra residues are left. Best of luck, Peter

[ccp4bb] Off topic: cDNAs

Hi, apologies for posting this off topic subject, but does anyone know where to get cDNAs for more exotic organisms? Specifically more fungal/protozoans such as K.lactis and C.glabrata. Thanks in advance, Peter

[ccp4bb] crystal growth

150mM NaCl, 5mM DTT, pH8.0. Thanks in advance for any thoughts on the matter. Peter Hsu

[ccp4bb] Postdoc position available at University of Washington Seattle

The Varani group at the University of Washington is looking for a postdoc to work in the area of RNA structural biology, primarily on RNA:protein and protein:protein complexes involved in RNA processing. Candidate must have a PhD in biochemistry or related fields, and a background in crystallog

[ccp4bb] Beginning crystallography text

Hi all, I haven't gotten past the phase of growing the crystal, but I'd certainly still like to learn the actual theories of crystallography. Can anyone recommend a good beginner to mid-level text on macromolecular crystallography? Thanks, Peter

[ccp4bb] Hampton Silver bullets

Hi all, I'm thinking about buying this screen for my group, but it is pricy. Has anyone had any luck with getting hits with these screens before when there were no hits before? Thanks

Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins

Buz, Have you tried putting on a solubility tag, such as GST? I've found some times using M9 to grow my proteins in can give more soluble protein, although yields are usually lower. Best, Peter

Re: [ccp4bb] bacillus protein expression

Amit, In line with that, you could also try a His-GB1 tag. NMR people often like to use this since it's a well folded, highly soluble and most importantly, small tag (~50 residues). Best, Peter

Re: [ccp4bb] protein degradation during concentration for crystallization trials

Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to near

Re: [ccp4bb] question about the zinc binding protein

Hi, I'm working with a possible zinc binding protein. Saw your post and was wondering, what is the proper pH range for zinc binding? Thanks, Peter