Re: [ccp4bb] Protein rapidly precipitates when off ice

Dear all, I will give my advice once again and hopefully you will discuss on it the next 2 days :P Chris might be worth trying a 2ndary or 3ary structure prediction tool to ensure that you do not have f.i. flexible ends. Disordered regions may lead to this kind of issues, as well. Personally I lov

Re: [ccp4bb] Protein rapidly precipitates when off ice

I'd try varying the pH independent of the theoretical pI, sometimes the real pI is very different. (I've worked on a protein with theoretical pI 9.2, real pI determined by iso-electric focussing 7.8). I'd also try limited proteolysis on the milky sample and see if you can solubilise it while a

Re: [ccp4bb] Protein rapidly precipitates when off ice

Hi, I was in Sung-Hou Kim's group when this work below was performed and published and I also tried it out on many occasions. Elegant piece of work and certainly worth trying. Aside from the suggestions of trying different pH and related optimum solubility screening and if higher salt and glycero

Re: [ccp4bb] Protein rapidly precipitates when off ice

Hi Chris, What is the theoretical pI of your protein? If it is around pH 7.5, you might try gel filtering your protein into a different buffer/pH combination. Try changing by at least 1 pH unit in either direction. If the pI isn't a problem, then you might try try solubility screening as outli

[ccp4bb] Stable Refinement as Low(ish) resolution - Follow up

Hi All, Thanks for all the useful comments. Buster gave me quite stable refinement and nice maps, so I've been working with that so far. I'll work through the other suggestions however, and see what gives the best results. Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash Univer

Re: [ccp4bb] Fine Phi Slicing

Jacob If you have a complete 360 deg data set and your sample is still alive, and you have a multi-axis gonio, I would recommend rotating the crystal about the beam (ideally by ~ maximum scattering 2-theta angle) and collecting again. This would record your blind region as well as moving the re

[ccp4bb] Protein rapidly precipitates when off ice

Dear CCP4BB Community, This week, I purified a nicely overexpressing protein by Ni-NTA followed by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration fractions to ~1 mL, transferred the spin filter to ice, and then collected 2 uL for measurement on the Nanodrop. Sadly, the prote

Re: [ccp4bb] Fine Phi Slicing

Hi Fred, fine slicing does not alleviate the count RATE limitation of photon counting detectors because fine slicing does not reduce the instantaneous photon flux on the detector when you cross the diffraction maximum. Fine slicing does help if you push the maximum counts per pixel per frame

Re: [ccp4bb] weird diffraction pattern

Dear Gerd, I wasn't really giving much attention to the poke between the ribs ;-) - for me the more serious matter was to see the merits of pixel detectors over CCDs made light of, as if they didn't really make much difference. If some people get carried away in the way you describe, we

Re: [ccp4bb] Fine Phi Slicing

I could be wrong here, but isn't the case that fine slicing is an option with a CCD and a necessity on a PAD b/c of dead time and/or counter dynamic range issues? (no current and/or former financial ties to any manufacturer) Fred 

[ccp4bb] Fine Phi Slicing

I thought there was a new paper from the Pilatus people saying fine slicing is worth it even beyond the original 1/2 mosaicity rule? I would think, actually, more gains would made by doing light exposures at, say, 1/3 mosaicity, collecting 360 deg, then shifting the detector in 2theta by a degr

Re: [ccp4bb] weird diffraction pattern

Dear Gerard, my "sound like a sales person" was meant as poking a little fun - nothing serious, of course. I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector in many ways. But, there is a lot of hype that this detector solves all-problem, for instance fine slic

Re: [ccp4bb] weird diffraction pattern

Thanks for spelling it out! Would that advice still hold if the mosaicity of the crystal is 0.7 degrees? (I know, I should go read the paper., but . . .) eab On 07/13/2017 03:00 PM, Gerard Bricogne wrote: Dear Gerd, I can assure you that I have no shares in Dectris nor any commecial conne

Re: [ccp4bb] weird diffraction pattern

Dear Gerd, I can assure you that I have no shares in Dectris nor any commecial connections with them. What I do have is a lot of still vivid memories of CCD images, with their wooly point-spread function that was affected by fine-grained spatial variability as well as by irredicible inaccura

Re: [ccp4bb] weird diffraction pattern

Dear Gerard, you sound like a sales person for Dectris. Fine slicing is perfectly fine with CCD detectors - it takes a bit longer because of the step scan instead of continuous scan. The read noise issue is often overstated compared to the sample induced scatter background. If for fine slicing

[ccp4bb] 2nd CryoEM sample preparation Workshop 2-4th Oct 2017 @ DLS - deadline for applications 6th august

Dear all we are pleased to announce a 2nd workshop aimed at newcomers to CryoEM for prepration and initial characterization of samples for single particle CryoEM experiments– full details in link below http://www.diamond.ac.uk/Home/Events/2017/cryoEM-Worskhop-October-2017.html the workshop will

Re: [ccp4bb] weird diffraction pattern

Hi Chenjun, Few suggestions from my side. Process the data with XDS and look into acentric intensity distribution (it indicates any twinning possibility). Run XTRIAGE and SFCHECK to understand any twinning or pseudo translation possibilities. Twinning can confuse the program and suggest you smalle

Re: [ccp4bb] weird diffraction pattern

- which may well be caused by your cryo-protection or flash-cooling procedure. I'd try to collect a few images at room temperature to see how good the crystals can be and if this procedure can be improved. To prevent overlaps, it may help to find a way to collect the data with the crystal rotatin

Re: [ccp4bb] crystallization optimization

Vaheh, one thing you can do is set up a microbatch-under-oil plate, varying protein against precipitant, with a nice big variation in both parameters. Then set up exactly the same experiment but with say 20 nl of seed-stock added to each drop. Hey presto that gives you the one dotted and two solid

Re: [ccp4bb] crystallization optimization

I’d be curious whether anyone has ever published an empirical phase diagram that looks like the one posted here, since I think real experiments have a lot more free parameters than those included in the phase diagram. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Og

Re: [ccp4bb] crystallization optimization

What I’m about to write should be referred as a question rather than an answer. However, it might also help to find the answer to crystallization question discussed here. The good old crystallization diagram so far for me was something that I’d look after successful crystallization story and fin

[ccp4bb] Major PDB FTP archive update to V5.0

*The model files in the PDB FTP archive have been updated to V5.0 of thePDBx/mmCIF dictionary .Both mmCIF and XML formats have been updated. These files were providedpre

Re: [ccp4bb] Granada Crystallization Boxes?

?If it weren't for the fact that trying to 3-D print translucent plastic is still problematic, you would think that this would be a perfect application of that technology. Diana ** Diana R. Tomchick Professor Departments of Biophysics and Bioche

Re: [ccp4bb] weird diffraction pattern

Dear Tang, I noticed that your diffraction images seem to have been recorded on a 3x3 CCD detector. With this type of detector, fine slicing is often discouraged (because of the readout noise), and yet with the two long cell axes you have, any form of thick (or only semi-fine) slicing would r

[ccp4bb] 2nd CryoEM sample preparation Workshop 2-4th Oct 2017 @ DLS - deadline for applications 6th august

Dear all we are pleased to announce a 2nd workshop aimed at newcomers to CryoEM for prepration and initial characterization of samples for single particle CryoEM experiments– full details in link below http://www.diamond.ac.uk/Home/Events/2017/cryoEM-Worskhop-October-2017.html the workshop will

Re: [ccp4bb] Granada Crystallization Boxes?

We probably would be interested in used ones. On Thu, Jul 13, 2017 at 4:18 AM, Patrick Shaw Stewart wrote: > > Gloria, would you be interested in used ones? I don't actually have any - > we threw them out a few months ago, I've just checked - but someone might > have some. > > Best wishes, Patr

[ccp4bb] AW: [ccp4bb] weird diffraction pattern

Hi Chenjun Tang, From the images you sent, it looks like your crystal suffers from lattice translocation disorder. See e.g. http://onlinelibrary.wiley.com/doi/10.1107/S0907444909025153/epdf Calculating a native Patterson and looking for strange peaks may give some hints what is going on. Depend

Re: [ccp4bb] weird diffraction pattern

Hi Jacob, I have tried seeding approaches but it didn't help. All best, Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Bal

Re: [ccp4bb] Inquiry - active and non-active state in alternatives

Dear Petr, I observed both open and closed protein conformations discretely disordered in the crystals of tyrosine phenol-lyase. In some cases, I could model two ligand conformations in the disordered active site, but the polypeptide chain could be modeled in the prevalent conformation only (fo

Re: [ccp4bb] crystallization optimization

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Re: [ccp4bb] Granada Crystallization Boxes?

Gloria, would you be interested in used ones? I don't actually have any - we threw them out a few months ago, I've just checked - but someone might have some. Best wishes, Patrick On 11 July 2017 at 19:04, Gloria Borgstahl wrote: > I have recently found out that these are no longer being manu

Re: [ccp4bb] weird diffraction pattern

You've got multiple lattices--try seeding approaches mentioned in a recent/current thread. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ??? Sent: Thursday, July 13, 2017 3:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] weird diffraction

Re: [ccp4bb] weird diffraction pattern

Hi, I'd need to see more processing stats to figure out your data issues. Streaky spots in certain directions can be indicative of anisotropy and/or lattice disorders. However, if your Rfree is above 0.5, it is likely that your molecular replacement solution is poor/bad/wrong. HTH, Dave --

[ccp4bb] weird diffraction pattern

hello everyone, I would like to seek your opinion on my crystal hits. I am working on a helicase of which the native structure is solved and the all solution statistics are fine. I am trying to crystallize and solve the structure of the protein/ssDNA complex. I recently got some hits from c

Re: [ccp4bb] Coot: changing scroll map keybinding

So, thanks to pointing by Paul, I have found the keybinding definition in "python/template_key_bindings.py" on coot's github. It is called "Step scrollable map number" with suggested key "M" Jan On 07/12/2017 10:33 AM, Jan Stransky wrote: Hi, I believe, I have seen somewhere a keybinding, wh

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

*Ahem* *ISOLDE Tristan Croll Research Fellow Cambridge Institute for Medical Research University of Cambridge CB2 0XY > On 13 Jul 2017, at 08:02, Tristan Croll wrote: > > This is more-or-less exactly the task I'm building ISODE for. It's early > days, still quite rough around the edg

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

This is more-or-less exactly the task I'm building ISODE for. It's early days, still quite rough around the edges and by no means complete, but if you have a machine with a decent GPU running a modern Linux distro, then it's easily available as a plugin to ChimeraX after installing the latest d