Re: [ccp4bb] About crystallization diffraction problem

Hi Anamika, 1) What is the conc of PEG3350, PEG6000 in the crystallization drop? If high enough, you may not need additional cryoprotectant. Just try freezing intact crystals without additional cryo and collecting data. 2) Grow the crystals in presence of ethylene glycol or glycerol (i.e. add c

Re: [ccp4bb] Refmac bond restraints across special positions?

Hi, > Is there a way to tell REFMAC that there are covalent bonds across asymmetric > units? Try this (example from 3gbi.pdb) for DNA: LINK PDC B 119 O3' DA B 125 1555 2555 1.61 LINK O3' DA B 125 PDC B 119 1555 3555 1.61

Re: [ccp4bb] high purity imidazole

Hi Vaheh, I believe this is the list of products you are looking for from 08/21/13? https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ccp4bb;aea5d439.1308 It has the Abs 280 nm of IMD from few different vendors. Regards, Debanu. -Original Message- From: CCP4 bulletin board [mai

Re: [ccp4bb] few questions about resolving new structure through MR

Hi Zhihong, The 3.5A diffraction could be due to many reasons: N- and C-term regions, interdomain linker possibly giving rise to molecular flexibility, quality of the particular crystals, cryo, purification, tags, etc. One thing to try is to run secondary structure predictions (or BLAST against

Re: [ccp4bb] 3-way obligatory associating proteins

An easy example is heterotrimeric G-proteins. In the termination cycle, G-alpha bound to GTP will hydrolyze the GTP, which then allows it to associate again with the G-beta/G-gamma dimer. Many membrane receptors (GPCRs, Chemotaxis, etc) can oligomerize and can be viewed like this, i.e., one dim

Re: [ccp4bb] Substrate/Ligand Induced Oligomerization of enzymes

Hi Shiva, There are some other enzyme systems activated by ligand induced oligomerization in addition to the ones on this list: 1) DNA polymerase dimerization on DNA binding 2) Restriction enzyme dimerization on DNA binding (an example is the type IIs FokI) 3) membrane enzyme receptors like rec

Re: [ccp4bb] list/library of most commonly co-crystallized ligands/solvents and/or their electron density shapes

Hi Jeremy, Given the large number of different kinds of ligands that are observed (either from crystallization, purification conditions or endogenous), I think it will be really difficult to assign just by visual and manual comparison with a standard set, especially for inexperienced crystallog

Re: [ccp4bb] unique structures

Hi Sandra, Yes, there are several ways of getting this information. Go to PDB website Advanced Search section. In the Query Type pull down, select All and then select "Remove Similar Sequences at 30% identity". You will have to select 30% cut off in the pull down menu. This will result in ~22

Re: [ccp4bb] solubility estimates for domains/structures?

Hi, Using a surface property analysis (3D structure is available), I think you can get a quantitative estimate by using PISA to find the solvent-accessible area and salvation energy and then comparing that to corresponding values obtained by using in your lab another protein for calibration (ma

Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins

Hi, Yes, it is worth trying. Nonionic detergents can be good for this. One example I know of and readily comes to mind is the use of 0.1% NP40 (Noniondet P40) in the stabilization of murine reverse transcriptase during purification (also helps toprevent precipitation), first described in: "Puri

Re: [ccp4bb] the lysozyme of membrane proteins?

The multidrug efflux transporter AcrB could be suitable including crystallization (both recombinant with His-tag and endogenous). Extracts, solubilizes, purifies and crystallizes in DDM, so it is fairly inexpensive to repeat in bulk compared to other detergents. Assays might be a bit complicated

Re: [ccp4bb] structural homology

Adding to the popular suggestions.. Check out the PSI Protein Model Portal (http://www.proteinmodelportal.org/), which allows submission to several servers including I-Tasser and Modeller as well as some informative tutorials on modeling workflow, etc. Also check out some other standalone serve

Re: [ccp4bb] Purify non-stable protein

Hi Theresa, The deletion probably led to a folding problem, making it susceptible to residual proteases. You might try the following: -Lysis and purification in the presence of 1mM EDTA, which can help to neutralize proteases in addition to the protease inhibitor, without significant leaching

Re: [ccp4bb] DDM

Hi Katarzyna, Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value is very low, at the extraction step you need to use a much higher concentration up to ~100x CMC. During different rounds of purification, you can bring the CMC level down to 1-2x CMC and even try deterg

Re: [ccp4bb] 96-well block storage

Hi, The polypropylene mat good for repeated sealing and reusing 96-well blocks than tape/film, to minimize evaporation. Hampton sells it: http://hamptonresearch.com/product_detail.aspx?cid=10&sid=88&pid=567 To address the mold problem locally (for the crystallization setup/room), the biocide so

Re: [ccp4bb] Ken Olsen, Founder of Digital Equipment Corporation, Died Sunday

Hi Dale, Thanks for posting this interesting piece of history. My non-PX initiation into PX was through DEC too. On a summer internship in 1996 after my junior year, I landed up at this institute which had just received their new DEC ALPHA2000. My project was to write up a C code (I had rece

Re: [ccp4bb] heavy atom soak

Hi Amit, For heavy atom phasing, you'll have to try all the things you can, limited only by supply of crystals, availability of heavy atoms (and considering heavy atom handling and waste disposal policies for solutions, tips, etc.) and time and effort. I had success with both short soaks (tri

Re: [ccp4bb] Suggestions for Reducing Protein Precipitation

Hi Matt, Check out the following paper and some screens available commercially based on these: Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug 26. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization

Re: [ccp4bb] Poor diffraction of eukaryotic membrane protein crystals

Hi Damon, In case your target oligomerizes to produce an assembly larger than 100 kDa, you may try to use the 100 kDa MWCO concentrator which will not only allow the detergent monomers to pass through, but also the micelles for most detergents used for membrane proteins. The 50 kDa one wil

Re: [ccp4bb] putting in methionines for SeMet crystals

Hi Amit, Based on the original analysis of M. Dayhoff (PAM matrix, Dayhoff substitution probability, Dayhoff, et. al 1978), introduction of Leu->Met would be the best choice for production of Se-Met derivatized protein. It would be best to consider a multiple sequence alignment of yo

Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal

Hi Rajakumara, You might try growing the crystals at say 25-30% of your PEG, it might be enough to cryofreeze without additional cryoprotectant if the current mother liquor is not enough. You may also try a serial soak in multiple steps increasing the cryo conc in steps of 3% or so to mak

Re: [ccp4bb] question about getting rid of model bias in refinement

Hi Bill, In PHENIX you can use the following commands for making different omit maps: A simple composite omit map can be made by: phenix.autobuild data=data.mtz model=coords.pdb composite_omit_type=simple_omit An iterative build omit map: phenix.autobuild data=w1.sca model=coords.pdb omit_box_

Re: [ccp4bb] high pI protein

Hi Mike, I would suggest that you run the protein sequence through the JCSG XtalPred server: http://ffas.burnham.org/XtalPred-cgi/xtal.pl (Bioinformatics. 2007 Dec 15;23(24):3403-5. XtalPred: a web server for prediction of protein crystallizability). This will give you a good idea i

Re: [ccp4bb] Parameter MAXSAVE exceeded

If you have phenix installed, you may also try: phenix.cns_as_mtz cns_file.cv http://www.phenix-online.org/documentation/reflection_file_tools.htm Regards, Debanu. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Miller, Mitchell D. Sent: Thursday, J

Re: [ccp4bb] Model ensemble for x-ray crystallography

Something to think about in using ensembles for refinement: Even in ultra high resolution crystal structures, usually only a single conformation for MC (maybe dual in some places) is seen and maybe 2-3 conformations for many SCs. Occupancies of these SCs can be adjusted during refinement with ev

Re: [ccp4bb] MolRep of coiled coils

Hi Thomas, MR of coiled coils can be quite tricky including considerations of being curved, etc. If conventional MR is failing (assuming you have tried different kinds of parameter and search model tweaks, you can also play around with the search thresholds in phaser), you may try the followi

Re: [ccp4bb] Model ensemble for x-ray crystallography

Hi, This idea was rejuvenated recently in the follg. work from 2007. I believe the article you are looking for is: Ensemble refinement of protein crystal structures: validation and application. Levin EJ, Kondrashov DA, Wesenberg GE, Phillips GN Jr. Structure. 2007 Sep;15(9):1040-52. Regards

Re: [ccp4bb] Off-topic structural genomics related question.

Hi, "Question posed in a different way - One experienced crystallographer is being supplied with crystals of proteins - how many can one solve in a year (40 hour weeks, with 5 week holidays)." Under your assumptions with slight modifications (median length ~365 aa, all Se-Met-mostly MAD, s

Re: [ccp4bb] Side chain chemical modifiers

Hi, You can use dimethylamine borane (DMAB) for reductive methylation of the surface exposed K's. You may also try NaBH4 or sodium cyanoborohydride but the first may be best. Don't know about D's and E's. Best, Debanu. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTE

Re: [ccp4bb] Thermofluor experiment

Hi, I did not see this particular instrument mentioned here so far: The FluoDia T70 from Photon Technology International, NJ, http://www.pti-nj.com/PlateReader/PlateReader.pdf The whole instrument comes with its own thermal cycler and 96-well fluorescence plate reader with appropriate excitat

Re: [ccp4bb] detergent concentration used for membrane protein purification

Hi Rongjin, During extraction from the membrane, typically 10xCMC (e.g. FC12) to 100xCMC (e.g. DDM) may be used, depending on target under study. During purification, it is recommended to bring this down to ~2xCMC. Theoretically, 1xCMC should be ok to keep the protein soluble. In practi

Re: [ccp4bb] elusive DNA density

Hi, >(1) Does the DNA density I saw in the cases where I use models with DNA for >MR/rigid body fitting only reflect model bias? If your DNA density is becoming poor as your refine, it is quite likely that the initial DNA density you are observing after MR is model bias due to inclusion of

Re: [ccp4bb] Modelling loop conformations

Hi, How big is the gap? Did you try lego_ca, lego_auto_mc or lego_loop in O. They will generate fragments for you to try from its database with good geometries. You can then select the one which you think fits best in the map. You can also try Xpleo: http://smb.slac.stanford.edu/~vdbedem/

Re: [ccp4bb] SFALL grid

Hi, Use keyword "GRID" in the script to set values: GRID The following general restrictions must be observed: >= 2 * HMAX + 1 >= 2 * KMAX + 1 >= 2 * LMAX + 1 In addition there are further space group dependent conditions as follows

Re: [ccp4bb] His tag does not bind.

Hi, This has been discussed often. You can look back through old posts or also look at troubleshooting pages in purification manuals that come with the some of the products like Qiagen Ni-NTA beads, etc. Briefly, you can try the following: a) Play around with loading rate, i.e., go down to m

Re: [ccp4bb] "Quick soak" method

Hi, I've had a couple of successful cases: 1) Uranyl acetate soak to get starting phases at 6.0A combined with a gold derivative at 3.8A. Both quick soaks of about 10-20 mins in 5-10 mM on crystals less than 100 microns. 2) Mercuric chloride soak of 10 mM for 10 mins to get starting phases

Re: [ccp4bb] Solvent content of membrane protein crystals

Hi, There are at least 4 methods to try to estimate amount of detergent in a membrane protein crystal which may lead to a more accurate estimation of asu content, etc. At the minimum, this may serve academic curiosity. At the best, if one obtains membrane protein crystals that do not diffract

Re: [ccp4bb] Depositing raw data

How about the Petabox: http://www.capricorn-tech.com/ "Capricorn Technologies was founded in 2004 and provides petabyte-class storage solutions for organizations worldwide. Capricorn's PetaBox technology grew out of a search for high density, low cost, low power storage systems for the world's

Re: [ccp4bb] Structure help

Hi Yanming, >>Story ends with my questions: >>1,It seems the 3rd copy non-exist or globally disordered, under this >>circumstance, can I say 'because of the 3rd molecule is globally disordered >>so >>>Rfree and R stay high (2mol/a.u with Rfree 30% R 27%, 1.8A data)? After finding the first

Re: [ccp4bb] follow up on model DNA-binding crystals?

protein-DNA complex system almost equivalent to the lysozyme model. Thanks, Debanu. -- Debanu Das, JCSG, SSRL. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Das, Debanu Sent: Wednesday, August 15, 2007 11:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb

Re: [ccp4bb] model DNA-binding crystals?

Hi Frank, The N-terminal catalytic fragment/polymerase domain of the MuLV Reverse Transcriptase is easy to express (large yield of soluble protein in E. coli expression, ~270 aa), purify (3 steps) and crystallize (overnight to couple of days with microseeding) with short stretches of DNA (8-1

Re: [ccp4bb] highly soluble proteins

Hi Yvonne, Were you able to check the polydispersity of your highly soluble protein sample by DLS? Non-monodispersity could result in reduced propensity for crystallization. Another thing you could then try is something like the "Optimum Solubility Screen" which basically involves buffer

Re: [ccp4bb] how to bring back the missing density for half of the structure

Hi Eric, If your Phaser results show a high Z-score (> 8) AND high LLG AND your solution packs without clashes AND refines (even though starting R/Rfree is high) AND reproduces density for the model portion AND produces some Fo-Fc density for the missing portion, most probably your solution is

Re: [ccp4bb] Protein-DNA complex for crystallization

Hi, You can try the following to improve your protein-DNA complex crystals: 1) Try HPLC purified oligos. You can get that from IDTDNA instead of the standard desalting form. Alternatively, if you have access to a reverse phase HPLC/column, if you order DNA with Trityl group on, run it on th

Re: [ccp4bb] Help with reducing crystal mosaicity

Hi Mary, There is another expt. you might look into after trying out the different cryoprotectants, small vs. large crystals, capillary mounts, etc. as suggested. Dr. Robert Thorne's group (from Mitegen & Cornell U.)) has been doing some studies on flash freezing procedures and effect o

Re: [ccp4bb] how to convert matrix to angle

I believe you can use lsqkab and DynDom. Get the direction cosines of the rotation axis w.r.t. the centroid, etc. and also get a graphical representation + some more numbers for angular rotation, etc. -Original Message- From: CCP4 bulletin board on behalf of U Sam Sent: Sat 7/7/2007 9:23

Re: [ccp4bb] how to convert matrix to angle

t 7/7/2007 8:21 PM To: Das, Debanu; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] how to convert matrix to angle Not that I want to open a can of worms here... but could someone explain what is meant with 'angle between domains'? Thx, br -Original Message- From: CCP4 bulletin

Re: [ccp4bb] how to convert matrix to angle

Hi, You can also use LSQKAB in CCP4 to get the angle between two similar domains. -Debanu. -Original Message- From: CCP4 bulletin board on behalf of Kay Diederichs Sent: Sat 7/7/2007 7:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to convert matrix to angle Jiamu Du schr

Re: [ccp4bb] L-alanosine

Hi Jackie, It seems that when the crystal structure of L-alanosine was solved, it was done from a solution made by adding conc. HCl (20 ul) to the compound in water (200 ul). See ref. in Acta Cryst., 1986, C42, 733-738. I don't know what the starting pH of the compound in water will be,

Re: [ccp4bb] Low resolution chain tracing

Hi Alexei, In case of very good main chain density like you have, you can decide on N->C directionality by looking for the slant of the C=O group in helices. As you go from N->C, the C=O has the tendency to slant upward. In case of beta sheets, it may be difficult to judge, but at this point,

Re: [ccp4bb] Program for colouring DNA

Hi Aaron, Since distortion from standard DNA conformation is measured by rigorously calculating a number of different parameters at numerous levels (base pair level, dinucleotide level, overall helical form, etc.), I believe your best bet would be to run your structure through 3-DNA (which al

Re: [ccp4bb] Program to evaluate RNA torsion angles?

This might be silly, but how could I know whether those deviations are outrageous or not? The following references may answer your questions: 1) Sims GE, Kim SH.

Re: [ccp4bb] Alternate space groups for MR in Beast

Hi Ed, I believe BEAST has been replaced by PHASER (which would allow you the option of testing both space groups with only a single input reflection file). I don't think there is any keyword in Beast which would allow you to do the same. Thanks, Debanu.

Re: [ccp4bb] microscope cameras

Hi Ursula, We pruhcased a Leica DFC model digital camera to go with our Leica microscope for imaging crystals. We are very happy with that choice. It may be more expensive that the Jenoptik one but I think it would be worth looking into. Thanks, Debanu. From:

[ccp4bb]

Hi Daniel, You can also refer to HATODAS, http://hatodas.harima.riken.go.jp/, where you can find suitable heavy atom compounds to use based not only on the sequence of your protein but also the crystallization conditions, as is your case. This is based on the following paper: Heavy-atom Databa