Re: [ccp4bb] protein expression in human cells

Hi Gloria, Another vote here for HEK 293 Expi or Freestyle. The off-the-shelf transfection reagents are super expensive, but I make my own (happy to share protocols with anyone interested) and we normally get a few mgs of protein per litre of suspension culture -- certainly enough for crystall

Re: [ccp4bb] protein expression in human cells

, 2020 7:10:06 AM To: David Briggs ; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] protein expression in human cells Dear colleagues, I wonder if there were a bit less controversial possibility. No matter if that was less efficient. Would there be an option of using human cell lines that do no

Re: [ccp4bb] Red trapezoids in Coot

Hi Joel, The red trapezoids are indicative of cis-peptide bonds that are exceptionally rare, and usually indicate incorrect tracing of the backbone (unless you have good enough data to unambiguously decide that a cis-peptide bond is correct). I'm afraid I don't know how to get rid of them, othe

Re: [ccp4bb] CCP4BB vs COVID19

For general interest, The Francis Crick institute in London is offering facilities to public health England, and over 300 Crick scientists have volunteered to help with testing. https://www.crick.ac.uk/news/2020-03-19_francis-crick-institute-offers-assistance-in-emergency-coronavirus-testing --

Re: [ccp4bb] disinfecting keyboards

Have you considered autoclaving the keyboards in between users? Or maybe autoclave the users? That'll work, right? -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs ___

Re: [ccp4bb] Molecular replacement problem

Hi Robert, Have you tried lower symmetry spacegroups? Maybe your crystal is 'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending to be orthorhombic. Zanuda can do this for you. https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html Good luck, Dave

Re: [ccp4bb] Commercial source of His-tagged protein

Hi Mark, NEB https://international.neb.com/products/p8112-tev-protease#Product%20Information

Re: [ccp4bb] electron density close Histidine side chain

Hi Samer, Did you use a His tag/Ni-NTA during purification? Sometimes Ni2+ ions leach off the Ni-NTA - maybe the two "ears" are accompanying waters? Ni-His co-ordination distance is pretty short (2-2.2Å - table 3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872550/#!po=0.69) and might a

Re: [ccp4bb] electron density close Histidine side chain

From: samer halabi Sent: 21 July 2020 11:08 To: ccp4bb@jiscmail.ac.uk ; David Briggs Subject: Re: [ccp4bb] electron density close Histidine side chain Hello, Thank you for your kind reply. If the distances are still less than 2.2Å, would coot and Refmac still consider

Re: [ccp4bb] File System Problems

Hi Sam, we had a similar issue with CCP4I2 on Linux in the last couple of weeks. Installing the very latest updates made the problem go away for us. I don't know if this Linux issue would also crop up in Windows, but what you describe seems similar - try to open a file selection dialogue and th

[ccp4bb] Diamond-II MX beamlines letters of support

Dear all, (Apologies for the slightly off-topic cross-post - hopefully, this will be of interest to all MX practitioners, not just Diamond light source users.) As Diamond User Committee MX representatives, we'd just like to remind you that the deadline for letters of support for Diamond-II beam

Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

A quick note regarding the code that Deepmind released for CASP13 (2018). It bears the rather important caveat that: "This code can't be used to predict structure of an arbitrary protein sequence. It can be used to predict structure only on the CASP13 dataset (links below)." Source: https://gi

Re: [ccp4bb] Protein elution in Size Exclusion

Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic

Re: [ccp4bb] Protein elution in Size Exclusion

column. > > Nian > > On Sun, Aug 28, 2011 at 9:24 AM, David Briggs > wrote: >> >> Following on from Roger's fine suggestions: >> >> 8. Your column is knackered. Can you see fine lines or cracks in the >> column?

Re: [ccp4bb] Finding a sequence motif with BLAST

Hi Jacob, SCAN PROSITE http://prosite.expasy.org/scanprosite/ will do precisely what you want. C-X-C-X-C-X-C or C-X-C-X-C would be the pattern using Prosite syntax. Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic =

Re: [ccp4bb] Vivaspin vs Amicon Ultra

Hi Jan, I've used both, and I've used them on membrane binding proteins, extracellular matrix proteins and intracellular proteins. I know of some cases where protein loss is very severe, but that varies on a case-by-case basis. A general observation is that proteins with which losses are bad, are

Re: [ccp4bb] Weird blob appears

I agree with Rafael, >From those pictures it looks like a sugar chain - maybe 2-3 saccharides in a row. HTH D David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk ==

Re: [ccp4bb] live streaming of ccp4 study weekend

Charles I would like to echo what Keitaro suggested - can the archieves of the talks be made available after ccp4we? Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchest

Re: [ccp4bb] off-topic: "special" format for multiple sequence (protein) alignment

I'm pretty certain that Jalview http://www.jalview.org/ can do that (you might have to set the user defined colours yourself). Dave. David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri.

[ccp4bb] Envelope Phasing.

Hi CCP4bb, I would like to ask about "envelope phasing" - specifically with SAXS data. There are papers (1) and tutorials (2) describing how this might be done, but I have also found comments on the ccp4bb, such as this one (http://www.proteincrystallography.org/ccp4bb/message11690.html) which ar

Re: [ccp4bb] Envelope Phasing.

lous difference data, though this > has yet to be shown in practice. Depending on the heavy atom this can > certainly help with the phase extension. > > > Was your question academic, or are you, like I was, stuck in low resolution > land with no phases? (I can certainly assist w

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

Hi Matt, This doesn't really answer your question directly, but sidesteps around the issue - I wrote a little something on this exact subject not so long ago - http://xtaldave.wordpress.com/2012/02/23/on-protein-crystallisation/ (You can ignore the first 5 paragraphs of intro - it was written with

Re: [ccp4bb] very informative - Trends in Data Fabrication

Trollus, Trollum, Trolli, Trollo, Trolli, Trollos, Trollorum, Trollis. David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk Webs : http:/

[ccp4bb]

Try adding ligand/receptor? Try an ortholog? (same protein, different species - slightly different protein sequence) On May 4, 2012 8:19 AM, "ruby singh" wrote: > Dear all, > > Im a PhD student who started working on Protein crystallization. Its been > years im trying to get any crystal of that

[ccp4bb] [OFF-TOPIC] Science is Vital.

Hi all. Apologies for the off topic, largely UK-centric posting. In a period of fiscal uncertainty in which most governments (USA, India, China, France, Germany) are actively increasing their investment in R&D, the UK government is seriously considering cuts of up to 25%, if press/analyst reports

Re: [ccp4bb] salt or protein crystals?

I agree - looks like small molecular diffraction. Try increasing delta-phi to catch more of the lattice to confirm - I often do a 5º image or two with the detector pushed as close as possible to check for salt diffraction when screening. The lack of low res (~15-20Å) spots around the beamstop is

Re: [ccp4bb] Structures determined: breakdown of methods

Rex, the PDB statistics page at the RCSB may contain the information you seek. http://www.rcsb.org/pdb/static.do?p=general_information/pdb_statistics/index.html HTH, David David C. Briggs PhD Father, Structural Biologist and Sceptic Uni

Re: [ccp4bb] Limited proteolysis affected by metal binding?

Hi Greg, I am not sure why you are so surprised! If the zinc is altering the conformation and/or folding of your protein, this might change the accessibility of trypsin cleavage sites, thus changing your limited proteolysis pattern. Eg: Metal-ion induced conformational changes in alkaline phosph

Re: [ccp4bb] diffraction of spherulites

Hi Stefan, It looks like a powder diffraction-type image. http://en.wikipedia.org/wiki/Powder_diffraction You can imagine that your spherulites are made up of many small crystals in random orientations, a bit like a powder - this means that rather than getting discrete diffraction spots as you w

Re: [ccp4bb] diffraction of spherulites

On 6 April 2011 11:29, Stefan Münnich wrote: > Hi Dave, > > thanks for your reply. Is there any chance of telling whether it is salt or > protein from the diffraction image? > > Stefan > > > On Apr 6, 2011, at 12:16 PM, David Briggs wrote: > >> Hi Stefan, >> &

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

I'll give my backing to the Nanodrop as well. I've used it in two different labs, for general yield checking use as well as prior to ITC experiments, and haven't found there to be any issues. That said, I've also used cuvettes, and I find that one the whole, cuvette-derived and nanodrop-derived m

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

y the > "protparam" tool at http://ca.expasy.org/cgi-bin/protparam > estimates the extinction coefficient given a sequence. > > > > David Briggs wrote: > ~~~ >> >> I wouldn't touch Bradford with a barge-pole. I've found it to be >> wildly inaccur

Re: [ccp4bb] Another paper & structure retracted

Perhaps paper and structure should be peer-reviewed independently, and only when both have been given the green-light should both be released, simultaneously. I don't see why we should be especially precious about reviewing structural data - we gladly hand functional data, protocols, etc to revie

Re: [ccp4bb] Enzyme-Ligand complexes

This paper might provide some inspiration! Crystallization of protein–ligand complexes A Hassell et al Acta Crystallogr D Biol Crystallogr 2006 vol. 63 (1) pp. 72-79 HTH, Dave David C. Briggs PhD University of Manchester E-mail: david.c.b

Re: [ccp4bb] where I have been going wrong in crystallization?

Hi Martyn, this recipe tends to work for me... Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.8 Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.8 Additional Reagents: Index Reagent 8, 22, 28, 31, 34, 40, 58, 59, 69, 86, 88 Mix equal amounts of lysozyme with reagent, incubate at 4 or

Re: [ccp4bb] DLS

Hiya. My two penneth on the Malvern Zetasizer. Simple, idiot-proof operation. Software is easy and intuative. Reports are customisable to give you what information you want. Problems easy to trouble shoot IMHO. Does DLS, SLS, melting point determination. After-care support is good from Malvern.

Re: [ccp4bb] 24-well sitting-drop plates

Sounds like you like to take a look at Art Robbins 24-4 (or is it 4-24?) intelliplate. In the Hampton catalogue. Hth Dave -- Delivered via an Android. On Feb 24, 2010 12:20 AM, "Jacob Keller" wrote: Dear Crystallogrpahers, what are the best 24-well sitting drop plates on the market? I have u

Re: [ccp4bb] crystallization of a macromolecular complex

Whilst I agree with Leo that mM is a much more satisfying or rigorous unit of concentration, the huge variability of solubility observed in proteins, and even between point-mutants means we may as well use gigatonnes / firkin, for all the difference it makes wrt comparing crystallisation trials.

Re: [ccp4bb] crystallization plates and robotics

Hi, I have used the Art Robbins "hydra" to transfer reagents from deep well block to crystallisation plate, prior to using a mosquito to setup the drops. Assuming they are still available (this was about 4 yrs ago) the hydra was robust and easy to use. HTH, Dave -- Delivered via an Android. O

Re: [ccp4bb] Butandiol as cryopotectant

2,3-BD users, What sort of concentrations are you using? Dave -- Delivered via an Android. On Mar 22, 2010 11:45 PM, "Steve Tuske" wrote: Hi Ulrike, 2,3-butanediol has worked very nicely as a cryoprotectant. In my hands it mostly seems to decrease mosaicity. I was able to switch from a thr

Re: [ccp4bb] X-Ray films

Terminator 3 - IIRC, a synchrotron strips the fluid-metal exoskeleton off of TX - that counts, right? -- Delivered via an Android. On Apr 15, 2010 9:07 PM, "Brock Schuman" wrote: Wrath of Khan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli wrote: > The documentary "N... -- --

Re: [ccp4bb] degradation of protein durring freez thaw

Hi, Obvious answer - don't freeze it. If you cannot set your crystallisation screens up straight away (the preferred option, IMHO), why not try leaving the protein at 4C overnight? Does it still degrade? Freezing & thawing a protein-protein complex not a good idea, I think. Hth, Dave -- Delive

Re: [ccp4bb] degradation of protein durring freez thaw

proteins I’ve worked on and it has only been for 98% of the cases examined. Cheers, tom -- *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *David Briggs *Sent:* Thursday, 22 April 2010 5:27 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re

Re: [ccp4bb] How to predict the secondary structure from a protein sequence..??

Hi Hussain, Psipred is a good a place as any to start: http://bioinf.cs.ucl.ac.uk/psipred/ Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk =

Re: [ccp4bb] Native Gel Theory and Practice

Dear Jacob, I know that this is not the answer you were seeking, but for a modest increase in the amount of protein required, a couple of analytical ultracentrifugation experiments would be able to determine stoichiometry and binding affinity for such a system. AUC has the added benefit of being a

Re: [ccp4bb] Finding best model for molecular replacement

I'll add another molecular replacement anecdote to the growing list: I like to generate some models using the "Phyre" server ( http://www.sbg.bio.ic.ac.uk/~phyre/ ) Feed the best .pdbs into Mr Bump. Go and get coffee. Come back and find a solution with post-refmac R/Rfree in the mid-30s. IMHO,

Re: [ccp4bb] Finding best model for molecular replacement

ordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 23 May 2010 21:27, Nathaniel Echols wrote: > On Sun, May 23, 2010 at 12:57 PM, David Briggs > wrote: >> >> I like to generate some mod

[ccp4bb]

Hammer time? On Jul 15, 2010 4:06 PM, "Badyal, Sandip K. (Dr.)" wrote: STOP

[ccp4bb] Crystal growth time lapse movies.

Hi all I'm doing a quick talk on crystals/crystallography for a "lay" audience in a couple of weeks time, and I'm looking for some time lapse movies / animated gifs of crystal growth. The one I was thinking of using was here: http://microgravity.msfc.nasa.gov/snell/vibration.html But that link i

Re: [ccp4bb] Crystal growth time lapse movies.

oettingen, Germany > Tel. +49-551-39-3021 or -3068 > Fax. +49-551-39-22582 > > > On Wed, 21 Jul 2010, David Briggs wrote: > >> Hi all >> >> I'm doing a quick talk on crystals/crystallography for a "lay" >> audience in a couple of weeks time,

Re: [ccp4bb] Suggestions for Reducing Protein Precipitation

Hi Matt, Have you tried changing the pH? Is it possible that at pH 8 you are at the pI of your protein (i.e. where it has zero net charge and is at its least soluble) ? I have also read a paper (can't find the reference right now) where the authors precipitated their protein by dialysing into wat

Re: [ccp4bb] Suggestions for Reducing Protein Precipitation

Hi all. I found that reference. :0) Acta Crystallogr D Biol Crystallogr. 2006 Jul;62(Pt 7):833-42. Epub 2006 Jun 20. Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches. Izaac A, Schall CA, Mueser TC. http://www.ncbi.nlm.nih.gov/

Re: [ccp4bb] Problem with getting protein-inhibitor complex structure

Hi Xiaopeng, There is no sign of extra density anywhere? Could the inhibitors be binding at a site other than the active site? Is the active site accessible from the solvent channels in the crystal? If not, best thing I can suggest is to try and co-crystallise protein with inhibitor - mix the two

Re: [ccp4bb] Practical MR advice

Hi Roger, I think your ideas are sound, but I would add some "prime-and-switch" density modification in resolve plus/minus ncs to try and improve the maps and cut down on phase bias. Hth, Dave -- Hand delivered by Androids On 1 Sep 2010 16:12, "Roger Rowlett" wrote: I am trying to find a MR

Re: [ccp4bb] Fancy crystal, poor diffraction

0 ext 2107 Email: [EMAIL PROTECTED] -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile

Re: [ccp4bb] Fancy crystal, poor diffraction

efully protein) happy. Surely the addition of a _reducing_ agent would _reduce_ your disulphides - i.e. break them. Dave Pete Pete Meyer Fu Lab BMCB grad student Cornell University -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.

Re: [ccp4bb] problem with anisotropic refinement using refmac - Sacrosanct R-free

l figure of merit = 0.9183 > ML based su of positional parameters = 0.0274 > ML based su of thermal parameters= 1.5420 > rmsBOND = 0.014 > rmsANGLE = 1.569 > > Thanks in advance, > georg zocher > > -- --- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile

Re: [ccp4bb] crystal friendly solvents that are useful for dissolving hydrophobic small molecules?

solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green -- ------- David Briggs, PhD. Father & Crystallogr

Re: [ccp4bb] protease cleavage sites

come Trust Sanger Institute Hinxton Cambridge CB10 1SA Work Tel: 0044 (0)1223 834244 ext. 7318 Cell No.: 0044 (0)7870 208280 === Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844 -- --

[ccp4bb] How to get rid of Membrane formed on hanging droplets?

ress: Street Address: NIEHS, MD F3-05 NIEHS, Building 101, Room F363 P.O. BOX 12233 111 T.W. Alexander Drive RTP, NC 27709 RTP, NC 27709 Tel (o): 919-316-4634 E-mail: [EMAIL PROTECTED] -- --- David Briggs, PhD. Father & Cry

[ccp4bb] Generate Random phase set.

ons). That's it. Thanks in advance, Dave -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should

[ccp4bb] Differentiating bound Mn & Ca.

one go about it? Cheers, Dave -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams

[ccp4bb] Differentiating bound Mn & Ca - Summary

or the other. Other ideas suggested closer to home include (micro)PIXE, and intact mass analysis - maybe. The obvious scan around the Mn K edge is still the definitive crystallography-based test.. Thank you very much for all your helpful suggestions. Dave -- ------

Re: [ccp4bb] R and Rfree

in the subsequent refinement, which one I should accept and go forward. Thanks. Sam _ Discover the new Windows Vista http://search.msn.com/results.aspx?q=windows+vista&mkt=en-US&form=QBRE -- ---

Re: [ccp4bb] How do CNS deal with the His NE2 atoms coordinated with Zn2+?

istance? Any help is welcome! Best regards, Deqiang Yao -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made Presiden

Re: [ccp4bb] Second moments of Z but not twinning

reciated. Thanks, Graeme -- ------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams

Re: [ccp4bb] B-factor & Space gr questions!

igid body refinement prior to positional refinement. Have the authors deposited the Structure factors? I would use EDS to check the maps out: eds.bmc.uu.se/eds/ thanks, Ibrahim HTH, Dave -- ------- David Briggs, PhD. Father & Crystallographer www.db

Re: [ccp4bb] The importance of USING our validation tools

t; clearly wrong. They can dump > blame on the technique and escape personal responsibility. This is what > upsets so many of us. > > It would be so refreshing to read in one of these responses "We were under > a great deal of pressure > to get our results out befo

Re: [ccp4bb] Tough ligand to bind?

in > > the future. > > > > Thank you in advance for your suggestion. > > > > Nian > > > > Dept of Biochemistry > > UT Southwestern Medical Center > -- --- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams

[ccp4bb] Rotation Axis visualisation.

? Cheers, Dave -- --- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the

[ccp4bb] Vary b-factors from a plane/point.

Hi everyone, I know that this has been discussed on the bb before now, but 20 pages of google search in, I have still come up with zip. I want to vary b-factors smoothly from either point or a plane. This is for pretty-picture purposes. I can then colour molecule by b-factor and get nice, pretty

Re: [ccp4bb] Rare syst. absences n P212121

Hi Enrique, Sounds like your crystal is trying to be P2(1)2(1)2(1) (or maybe something higher, given the 4n) , but just not quite making it - maybe the cryoprotection or freezing has caused shifts within the lattice. Have you taken any room temperature shots? Other than that, have you tried just

Re: [ccp4bb] more on dimerization interface analysis

Hi Andreas, This maybe a little ott, but the Rosetta suite of modelling programs will do docking with some minimisation and side chain optimisation. You can also enforce symmetry. http://www.rosettacommons.org/ http://depts.washington.edu/bakerpg/ It is free to academics and runs on most platfor

Re: [ccp4bb] insoluble ligand

The review cited below is a great source of ideas to help you get your ligand in your crystals Hassell, A et al Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):72-9. Crystallization of protein-ligand complexes. Hopefully it may help you! Dave On 11/12/2007, Schubert, Carsten [PRDUS] <[EM

Re: [ccp4bb] protein precipitated when they formed a complex

Hi Jerry, have you tried tinkering with the pH? I have had similar situation, where two normally soluble proteins precipitated upon mixing at pH 7.5, my "stock" buffer. Altering the pH showed that the complex that was only soluble at pHs lower than 6. Looking at your pIs, it strikes me that in b

Re: [ccp4bb] WAS - Occupancy refinement... NOW - shameless promotion of facebook event.

Whilst the joys of Facebook are fresh in our minds... http://www.facebook.com/event.php?eid=6213885939 D On 17/12/2007, Gerard DVD Kleywegt <[EMAIL PROTECTED]> wrote: > > > I thought that I would never have to disagree with both Eleanor and > > Tassos in the same email, let alone risk being burn

Re: [ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?

Covalent modification?? Bias in 2fo-fc map? Can you show us a pic of offending density? Dave On 28/12/2007, Brenda Patterson <[EMAIL PROTECTED]> wrote: > > Hello, > > I am fairly new to this lark so please forgive me if this question is > unclear, > but it is really puzzling me. > > I have used

Re: [ccp4bb] crystallization robot

With regard to the Phoenix: I'm not sure why you've had these problems with the robot, We use 100+100ul regularly and the CryCam has little or no problem scanning in the drops. Maybe it needs a little TLC to get it properly aligned. 12mins seems a little too long for a 3x96 plate - I'm sure with

Re: [ccp4bb] crystallisation robot

I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. D

Re: [ccp4bb] combine incomplete data sets

Hello Yadong, This is what I would do in your position. 1) Integrate everything in MOSFLM 2) Enforce consistent indexing using POINTLESS (unless I am mistaken, there are alternative origin(s) in p321) http://www.ccp4.ac.uk/dist/html/alternate_origins.html ftp://ftp.ccp4.ac.uk/ccp4/6.0.2/prereleas

Re: [ccp4bb] combine incomplete data sets

Oops. My link & terminology were a little wayward... I knew what I meant, but the incorrect link may have proven misleading. But the answer essentially remains the same: 1) Integrate everything in MOSFLM 2) Enforce consistent indexing using POINTLESS (unless I am mistaken, there are alternat

Re: [ccp4bb] protein expression problem

Hi Chen, You could try adding some detergent or other solubilising agent (eg NDSBs) to your buffer. Have you tried other pHs? If you are sat near to or on the pI of your protein, it will be at its least soluble and more likely to aggregate. I've had protein behave like yours at pH 7.5 but behave p

Re: [ccp4bb] salt sensitive complex

Hi Jerry, to summarise your problem, using (close to) physiological buffer, SPR and ITC give you different results, you get different results in different salt strengths and to add to your misery, the proteins precipitate at low salt concentrations when mixed to together. Ok. Given the above, yo

Re: [ccp4bb] Characterization of common salt crystal forms?

Hi Joe, I've known most salt crystals in Phosphate - and I think most people are weary of phosphate. Also, Calcium Sulphate is a fairly common one, esp if your buffers are titrated with sulphuric acid. Fluoride Ions are also prone to form salt crystals with transition metal ions. HTH, Dave On

Re: [ccp4bb] The Lawnmower Man

Wow, that is like, so last week(!). I'm using COOT though a wireless interface to a head-up-display mounted inside my contact lenses, controlled via a neural lace* embedded directly into my parietal lobe. I can refine and rebuild my structure whilst doing minipreps, having a coffee, in the shower

Re: [ccp4bb] motif search

Hi Sean, You could try a "scan prosite" search and tell it only to search the pdb http://www.expasy.org/tools/scanprosite/ You would enter a search pattern such as D-A-V-E and it allows you to look for exact matches. You can also specify to look for homologues, like [DE]-A-[VLI]-[DE] This woul

Re: [ccp4bb] which concentrated salt has lowest vapour pressure?

I'm not entirely sure this paper will answer your question, as I can't access the full article at home, but I seem to remember it may contain what you seek. It's certainly worth giving it another airing... http://scripts.iucr.org/cgi-bin/paper?S0907444905002726 Acta Cryst. (2005). D61, 490-493

Re: [ccp4bb] How to dissolve a hydrophobic peptide?

This might help... Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL 3rd, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM. Abstract Crystallization of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2007 Ja

Re: [ccp4bb] Density Joining Cysteine and Lysine Side chains

Hi Andrew You don't say what your protein is crystallised in, or has seen during purification, but maybe S- MERCAPTOCYSTEINE might fit? (search for CSS in MSD CHEM) Do you have lower wavelength data that you might get a sulphur anomalous signal to check this? S-methyl Cysteine might fit as well -

Re: [ccp4bb] Density Joining Cysteine and Lysine Side chains

...by lower wavelength, I mean longer wavelength... or lower energy. Take your pick. Dave 2008/5/22 David Briggs <[EMAIL PROTECTED]>: > Hi Andrew > You don't say what your protein is crystallised in, or has seen during > purification, but maybe S- MERCAPTOCYSTEINE might fit?

[ccp4bb] Pointless Error.

Morning all... I've just downloaded the newest version of pointless, 1.2.15, using the mac osx intel binary. When I try and run it through the gui, it seems that my child gets hit by some sort of bus error: *** * Informatio

Re: [ccp4bb] Pointless Error.

e now and it is > working fine - take a look at the MRC ftp site to see if you can get the > same binary: > > ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre > > Cheers, > > Graeme > > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf

Re: [ccp4bb] Pointless Error.

; > That one works fine on my machine - intel mac, 10.4.11 as is yours - > you should be fine then. Now I think on when I asked about this a > leopard was involved, so I ended up using this. > > Cheers, > > Graeme > > 2008/5/23 David Briggs <[EMAIL PROTECTED]>: >

Re: [ccp4bb] Protein binding to Zn and Ca

Hi there. Clostridial Alpha toxins (phospholipase Cs that cause gas gangrene) bind 2-3x Zinc in the catalytic site, and 2-3 Ca in the membrane binding domain. See 1ca1, 1olp, 1qmd and others. Dave 2008/6/13 Neeraj Kapoor <[EMAIL PROTECTED]>: > Hi all, > I recently came across a question ab

[ccp4bb] Molecular Dynamics for dummies.

Hi everyone. I have a slightly off topic question I hope someone can help with. I have a structure of a wild type domain, which binds metal ions. Certain mutations in chelating residues cause changes in the apparent affinity for said metal ions. As I have (so far) failed to crystallise the mutan

Re: [ccp4bb] DLS options?

I've used variations of the Malvern instrument at two positions now, and I have to say I've never had a problem with them. Yes, I believe it was designed to be a non-biological instrument, but I have to say it does a good job of DLS and SLS on proteins from 7-200KDa (in my experience) in a cuvette

Re: [ccp4bb] Na Acetate Buffer

Hi y'all. One nice cheat is to get a groovy little web server to do the work for you: http://www.liv.ac.uk/buffers/ Enter your requirements and you'll get a nice little recipe given back. Dave 2008/7/22 Nadir T. Mrabet <[EMAIL PROTECTED]>: > I bet it is more difficult to adjust a pH-meter than

Re: [ccp4bb] MR problem --molrep

Hi there, I think 55% completeness is insufficient - you really need to collect more data. Take a look at this movie from James Holton's website: http://ucxray.berkeley.edu/~jamesh/movies/osc.mpeg Stop the movie at 55% and check out how lousy the maps are compared to 95-100% - and I think these

Re: [ccp4bb] jokes out of science for science community

Whilst the science itself might not have them rolling the in aisles, you can at least try and have fun whilst doing it. http://www.phdcomics.com/comics.php?f=1056 After all, if you can't laugh at triplet-phase invariant probability distributions, then what can you laugh at. 2008/8/30 William G

Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard

Not wishing to pooh-pooh attempts to get Apple to right their wrongs (I am an Apple user myself) but I get along perfectly well without stereo, thank you very much. I find that depth-cue/fog is a sufficient cue for me to determine the "3-dimensionality" of what I am viewing on my 2-dimensional moni

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