[ccp4bb] Ramachandran statistics and referee responsibility
Dear all, I have two questions. I would like to find out the community consensus of (1) best practices in refining against lower resolution data (~4 angstrom) to achieve the best model, and also (2) what manuscript referees should ask for in this regard. One might encounter a hypothetical situation where standard refinement approaches gave a model with poor Ramachandran statistics. Imposing Ramachandran restraints gave a model with improved Ramachandran statistics but at the expense of higher Rfree. I would expect that the model with better geometry is probably more reliable, and wonder if this is the general consensus view? I also wonder should a referee be the geometry police, or should authors/depositors have discretion to submit/publish the model they prefer so long as refinement protocol is accurately described and weaknesses such as poor Ramachandran statistics are evident in the presentation of the data and not concealed? Thanks for your input! Evette Evette S. Radisky, Ph.D. Associate Professor and Consultant Department of Cancer Biology Mayo Clinic Cancer Center _ Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471
Re: [ccp4bb] For stabilizing protein-protein complex
I agree with Engin’s suggestions. Our group crystallized a complex of mesotypsin with BPTI, where we measured an inhibition constant (approximating Kd) of 14 micromolar, by mixing the two pure proteins together in 1:1 stoichiometry. Actually we do that for a lot of our complexes with higher affinity also. It is definitely worth a try. http://www.jbc.org/content/283/7/4115.full Evette Evette S. Radisky, Ph.D. Associate Professor and Consultant Department of Cancer Biology Mayo Clinic Cancer Center _ Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Engin Özkan Sent: Tuesday, January 31, 2017 1:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] For stabilizing protein-protein complex Not observing a complex on a gel filtration run for a week complex (micromolar) is not necessarily unexpected. It all depends on your protein concentrations, the dissociation kinetics (remember, a gel filtration run is not an equilibrium experiment - it dilutes and separates your sample), etc. I would make sure that the SPR results are believable and sensible: if the affinity is really weak, I'd expect very fast kinetics - most weak interactions come with faster association and dissociation. As a result, in this affinity range, you can't fit kinetics data in SPR, and can only do an Langmuir isotherm fit to your equilibrium binding response values. Also, you should check your expected Rmax values (based on how much you captured on the chip), and make sure that your responses don't exceed the theoretical values. Otherwise, you are just observing non-specific binding. Similarly, if your responses are way too low compared to the expected, your protein on the chip might be mostly inactive - again, not good. If everything checks out, just mix your two proteins at the right stoichiometric ratio, concentrate a lot, and go ahead with crystallization (if that's what you want). Engin On 1/31/17 10:05 AM, sanjeev kumar wrote: Dear all, I am trying to stabilize a protein-protein complex. Our SPR study indicates it is having micro molar dissociation constant. I tried to purify both the molecule in complex form with size exclusion chromatography (mixed both the protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt observed formation of complex as both the molecule eluted at their respected elution volume. Please suggest me to get a better way to achieve the complex and if anyone gives idea about what is the good cross-linker I can use. Suggestions are highly appreciated. Thanks best sanjeev kumar, PhD Purdue University West Lafayette Indiana
Re: [ccp4bb] Cleaved peptide density!
We had a similar situation with a catalytically dead serine protease. Initially I was excited to think we might be seeing residual catalytic activity of the mutant enzyme on a highly specific substrate; however, the activity turned out to result from contamination with a very small amount of wt enzyme, likely as a result of using the same affinity column to purify both. When we incubated the enzyme preparation with PMSF (an inhibitor that covalently modifies the catalytic serine and would not have affected the mutant), we eliminated the residual activity of the enzyme preparation. However, in our case we never were able to get crystals with the intact substrate, which apparently was not compatible with our crystal form. Is there a covalent inhibitor of your cysteine protease that you could use to pre-treat your enzyme, to see if this eliminates the activity? If so this might help distinguish between residual activity of the mutant vs. contamination with wt enzyme. Good luck! Evette Evette S. Radisky, Ph.D. Associate Professor and Senior Associate Consultant Department of Cancer Biology Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/labs/proteases-cancer/ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Monday, April 20, 2015 2:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
Re: [ccp4bb] need some suggestions for crystallization
Bovine trypsin works well. You can buy it pretty cheap from Sigma and it crystallizes without further purification, within a week. Crystals diffract to 1.1-1.3 A and are quite robust to handling and soaking. Conditions that I used are described in this ref: http://www.pnas.org/content/103/18/6835.long I would avoid commercial preps of porcine elastase; I messed around with that one around 10 years ago using many reported crystallization conditions and suppliers but never got crystals. Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Roberts Sent: Monday, February 04, 2013 11:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] need some suggestions for crystallization So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave
[ccp4bb] Somewhat OT: question of professional courtesy
Dear bb, This morning as I scanned an accepted manuscript from a well-respected-but-not-particularly-glamorous journal that publishes many macromolecular structures, I came across a brief mention of homology and rmsd with a published structure listed by PDB accession number, but no citation of the primary reference for this structure. (OK, so I wouldn't have noticed or cared had it not been one of mine.) The paper did not have a lot of references, so it was not due to limitation in the number of refs permitted. I have always thought it a matter of professional courtesy to cite the appropriate reference when one uses and mentions a structure from the PDB, but as I think back, I realize no one explicitly told me this-- it is just an assumption that I made. Maybe I am the one with unrealistic expectations here? Is there a general consensus among crystallographers on this practice? Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372
Re: [ccp4bb] Somewhat OT: question of professional courtesy
Well, I guess that would be because when my CV and credentials are reviewed by e.g. study sections of funding agencies, institutional committees making decisions affecting my promotion and academic advancement, potential future employers, etc., many will look at metrics such as h-index and the like, based upon citations of publications rather than deposited structures. I am not saying that this is the way it would be in a perfect universe, but this is the way it is. Evette -Original Message- From: Tim Gruene [mailto:t...@shelx.uni-ac.gwdg.de] Sent: Wednesday, July 25, 2012 9:15 AM To: Radisky, Evette S., Ph.D. Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Somewhat OT: question of professional courtesy -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Evette, the PDB lists the citation when you enter the PDB-ID in the search mask of any of the web-interfaces, which is much easier for the reader than typing the information from the list of references, i.e. all information is in the article by mentioning the PDB-ID. Why do you consider it a matter of courtesy to re-cite the structural work? Cheers, Tim
Re: [ccp4bb] Somewhat OT: question of professional courtesy
Yes, I think this is reasonable. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R. M. Garavito Sent: Wednesday, July 25, 2012 9:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Somewhat OT: question of professional courtesy Evette, I think the primary issue is what kind of analysis was being reported on. That is what I look for when I review a manuscript. If the authors are doing a broad structural analysis (homology of TIM barrels, X-ray refinement protocols, etc.), I wouldn't expect citations beyond stating the PDB entries used. However, if this was a primary structural analysis of a macromolecule, I would expect that a discussion of the structural comparison would include references to earlier work(s) on related molecules, but I have seen this happen where a group reinvents the wheel (sometimes rather badly) because they don't take the time to look at the literature, just a DALI run and a PDB search. It is just bad science not to discuss what earlier researchers have done to put your work in context. Just my 2 cents worth, Michael
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
As I recall, I used to wear latex gloves, add extra padding to the handle end of the wand (and other tools) with bits of tubing, and take my tools out of the cold room every 20 min or so to de-ice and dry them. I didn't really have a choice about the cold room because it was the only place my crystals would grow, and my crystallization solution was 20% isopropanol which was too volatile to work with outside the cold room anyway. Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, Juergen Sent: Friday, July 13, 2012 8:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) This is a very interesting topic I have to say. But what I missed in this discussion is the pain you go through when freezing in the cold room. As the name implies it's supposed to be cold (most of the times). But that's not too much of an issue as you can dress up accordingly. The problem I always had was freezing up of the advertisement Hampton Magnetic Wand /advertisement and icing up towards your fingertips after some time when moisture from the cold room condenses and freezes. I hate wearing gloves when handling crystals so there was not much of a skin protection. How do you guys solve this problem ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
Several have mentioned harvesting in the cold room to reduce evaporation. I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated. I've also hesitated to recommend it to trainees in my current lab for the same reason. Does anyone have solid information on this? I would like to be convinced that such fears are unfounded ... Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Thursday, July 12, 2012 2:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo for high salt crystal We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in water, so I would not expect cryoprotectant concentrations of glycerol or glucose to cause precipitation (We can save cryoprotectant solutions of at least 2 M ammonium sulfate indefinitely). How are you introducing cryprotectant? We use one of two methods: 1. Fish the crystal out of the mother liquor and place into artificial mother liquor with the same composition as the well solution + cryoprotectant. For glycerol or other liquids, you have to make this from scratch. For glucose, we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix well of course before use. Gentle heating in a block or sonication will help dissolve the glucose. 2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop the crystals are in. You can do this all at once, or in stages, keeping the drop hydrated by placing the hanging drop back in the well between additions. If your drops are drying out during crystal harvesting (very possible in dry conditions), you might try harvesting in the cold room, where evaporation is slower. We often have problems with crystal cracking and drop-drying in the winter months when the humidity is very low indoors. The cold room is usually humid enough and cold enough to slow evaporation to allow crystal harvesting. (I hate working in the meat locker, though.) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 7/12/2012 12:55 PM, m zhang wrote: Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks, Min From: jim.pflugr...@rigaku.com To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 + Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 50% saturated in reservoir. You will have to TEST these. See also this webinar on cryocrystallography which shows how to make these solutions: http://www.rigaku.com/node/1388 You could also try high salt solutions with similar technique. Good luck! Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [mzhang...@hotmail.com] Sent: Tuesday, July 10, 2012 11:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryo for high salt crystal regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks, Min
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
Thanks much. It helps a lot. On Jul 13, 2012, at 5:31 PM, Henry Bellamy hbell...@lsu.edu wrote: liquid N2 expands about 600 fold to RT gas. The minimum O2 concentration is 19% (per OSHA I think) so if the amount of vaporized N2 is greater than 2% of the cold room volume you could have a problem. One has to account for the possibility that the Dewar will break or be tipped over and all the N2 will be vaporized at once. HTH Henry Bellamy
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
My cold room is also too small for comfort, but I think your method could work in a pinch. Thanks! Evette Radisky, PhD Mayo Clinic Cancer Center Griffin Cancer Research Building 4500 San Pablo Road Jacksonville, FL 32224 tel: 904-953-6372 fax: 904-953-0277 On Jul 13, 2012, at 5:52 PM, tom.p...@csiro.au tom.p...@csiro.au wrote: Hello Evette, It will depend on the circumstances- how big is your cold room, how much liquid N2 you have in there, etc. You can actually calculate the percentage of N2 (or correspondingly, O2) in the air if all of the liquid N2 were to boil off at once. If the O2 goes below 20% it will feel like you are at elevation and if it goes below 19% it isn't good (I believe most oxygen sensors used for this kind of application alarm if it goes below 20% and then alarm strongly below 19%). As you, we generally avoid cryo-cooling crystals in the cold as we have a small cold room and no real ventilation- just a blower. If we use liquid N2 in there, we keep the door open and have someone stand outside. There is no warning with N2- you just fall unconscious. Best of luck, tom Tom Peat Biophysics Group CSIRO, CMSE 343 Royal Parade Parkville, VIC, 3052 +613 9662 7304 +614 57 539 419 tom.p...@csiro.au From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky, Evette S., Ph.D. [radisky.eve...@mayo.edu] Sent: Saturday, July 14, 2012 7:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) Several have mentioned harvesting in the cold room to reduce evaporation. I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated. I’ve also hesitated to recommend it to trainees in my current lab for the same reason. Does anyone have solid information on this? I would like to be convinced that such fears are unfounded … Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Thursday, July 12, 2012 2:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo for high salt crystal We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in water, so I would not expect cryoprotectant concentrations of glycerol or glucose to cause precipitation (We can save cryoprotectant solutions of at least 2 M ammonium sulfate indefinitely). How are you introducing cryprotectant? We use one of two methods: 1. Fish the crystal out of the mother liquor and place into artificial mother liquor with the same composition as the well solution + cryoprotectant. For glycerol or other liquids, you have to make this from scratch. For glucose, we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix well of course before use. Gentle heating in a block or sonication will help dissolve the glucose. 2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop the crystals are in. You can do this all at once, or in stages, keeping the drop hydrated by placing the hanging drop back in the well between additions. If your drops are drying out during crystal harvesting (very possible in dry conditions), you might try harvesting in the cold room, where evaporation is slower. We often have problems with crystal cracking and drop-drying in the winter months when the humidity is very low indoors. The cold room is usually humid enough and cold enough to slow evaporation to allow crystal harvesting. (I hate working in the meat locker, though.) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edumailto:rrowl...@colgate.edu On 7/12/2012 12:55 PM, m zhang wrote: Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks, Min From: jim.pflugr...@rigaku.commailto:jim.pflugr...@rigaku.com To: mzhang...@hotmail.commailto:mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 + Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated
Re: [ccp4bb] Is there a protein that it could interact with different proteins by the same part?
This is very common among the small GTP-binding proteins of the Ras superfamily. For an interesting analysis (although doubtless there are lots of more recent examples) you might look at: Corbett and Alber, TIBS 26 (12) 710-716 (2001) Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yamei Yu Sent: Tuesday, August 23, 2011 9:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Is there a protein that it could interact with different proteins by the same part? HI all, We know one protein can interact with different partners by different domains or different parts. Is there a protein that it could interact with different proteins by the same part (maybe the same part but in different conformations?)? Thank you in advance!! yamei
Re: [ccp4bb] what to do with disordered side chains
Just create a new tag, say _atom_site.imaginary_site, which is either true or false for every atom. Then everyone would be able to either filter out the fake atoms or leave them in, without ambiguity or confusion. Aside from being a binary rather than continuous parameter, how exactly does this suggestion differ from the occupancy column of the pdb, that we already have? I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372
Re: [ccp4bb] SUMMARY: finding I/Sigma(I) from HKL Scalepack
Thanks very much for all of the helpful suggestions! The most useful solution for us was a python script provided by Ed Pozharski at the ccp4wiki link below, which reads a .sca file and produces a table with both I/sigma and I/Sigma for a user-defined number of shells. http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Calculate_av erage_I/sigma_from_.sca_file Other suggestions are listed below: From Ed Pozharski: ctruncate outputs the table with Mn(F/sd) (which will be twice Mn(I/sd)) when it does anisotropy analysis From Phil Jeffrey: I personally report I/sig(I) but wrote my own program to do it from the .sca files. From Paul Smith: I have a perl utility that does much of what you would like. It will run scalepack for you iteratively until the number of rejections converges, or it will parse scalepack output. The output has I/sigI by shell gleaned from the scale log with the same resolution bins used in scaling. The usage for parsing is autoscale.pl -e scale.log From Wladek Minor: Click report on the top panel and you will get all relevant statistics that are necessary for publications. From Graeme Winter: You can still get this analysis with Scala even after scaling with scalepack. If you output the measurements unmerged (no merge original index) you can convert them to MTZ using pointless, then remerge the data as follows: scala hkiin from_pointless.mtz hklout merged.mtz eof run 1 all scales constant sdcorrection noadjust norefine both 1 0 0 cycles 0 eof (pointless -c scain ... - you will also need to assign the cell and symmetry) This will just remerge the measurements and give you the usual merging analysis from Scala. Very useful. Same trick also works with data from XDS/XSCALE. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Radisky, Evette S., Ph.D. Sent: Monday, November 01, 2010 4:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] finding I/Sigma(I) from HKL Scalepack Dear all, I have previously used SCALA for data reduction, and in publications and pdb depositions, reported the Mn(I/sd) output from SCALA for the whole data set and for the highest resolution shell. We now have some data that has instead been reduced using the HKL suite, and I am confused about how to find the value that would be equivalent to Mn(I/sd) from SCALA. For I/Sigma(I) I've been advised by a colleague more familiar with HKL to manually calculate from average I divided by average error (sigma). As pointed out in a previous ccp4bb thread, this would give me I/Sigma(I), which is not the same as I/Sigma(I). Two questions: (1) Is this I/Sigma(I) what is generally reported in the literature for data processed with the HKL suite? (2) Since I would also like to know the Mn(I/sd) by shell anyway so that I can compare to previous data sets, is there a way to extract this value from the scalepack log, or is there a simple reflection file analysis utility that could read the .sca or .mtz file to extract this information? Thanks for any clarifications or suggestions! Evette Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372
[ccp4bb] finding I/Sigma(I) from HKL Scalepack
Dear all, I have previously used SCALA for data reduction, and in publications and pdb depositions, reported the Mn(I/sd) output from SCALA for the whole data set and for the highest resolution shell. We now have some data that has instead been reduced using the HKL suite, and I am confused about how to find the value that would be equivalent to Mn(I/sd) from SCALA. For I/Sigma(I) I've been advised by a colleague more familiar with HKL to manually calculate from average I divided by average error (sigma). As pointed out in a previous ccp4bb thread, this would give me I/Sigma(I), which is not the same as I/Sigma(I). Two questions: (1) Is this I/Sigma(I) what is generally reported in the literature for data processed with the HKL suite? (2) Since I would also like to know the Mn(I/sd) by shell anyway so that I can compare to previous data sets, is there a way to extract this value from the scalepack log, or is there a simple reflection file analysis utility that could read the .sca or .mtz file to extract this information? Thanks for any clarifications or suggestions! Evette Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372
Re: [ccp4bb] protein monitoring
Try a Bradford-type assay, scaled down to microplate format. You can buy reagents pre-made; Pierce makes a good one . It is fast-- you pipet 10 microliters or so from each chromatography fraction into a well with the detection reagent, and wait 5 minutes. If protein concentrations are moderate to high in your peaks, you won't even need to use a plate reader; the fractions with protein will be quite visibly blue against a white background. You would probably want to check the MW of your protein peaks on a gel anyway, but at least you won't need to run lanes with a lot of fractions containing no protein. Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sollepura Yogesha Sent: Friday, May 28, 2010 12:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein monitoring Dear All, I have expressed 30-40 aa region my protein fused to GST. I subjected it to precision protease cleavage. On the gel I can see the band. When I looked for ProtParam in expasy it shows that my peptide doesn't have Extinction coefficients as there are no Trp, Tyr or Cys in the region considered, your protein should not be visible by UV spectrophotometry. I need to separate GST from the cleavage mixture. How can I monitor my peptide during FPLC and after that. AA composition is Ala (A) 8,Arg (R) 2, Asn (N) 3, Asp (D) 2, Gln (Q) 2, Glu (E) 2, Gly (G) 4, Ile (I) 1, Leu (L) 4, Lys (K) 4,Phe (F) 2, Pro (P) 2, Ser (S) 5, Thr (T) 5, Val (V) 3. I am looking for some suggestions Thanks in advance Yogi
[ccp4bb] SUMMARY: off-topic: selective reduction of surface cysteine
Thanks to all for your help and suggestions. We are still working out the best protocol for our specific protein, but I list here the suggestions received, since they may prove helpful to others as well. 1. I assume that your native (12 Cys) protein runs as monomer on non-reducing SDS-PAGE and on native conditions, right? If your 13 Cys protein is still correctly folded, the dimerisation has happened spontaneously, but can be competed off by a proper redox system, e.g. oxidized and reduced glutathione: GS-SG, GSH; I would start with 1 mM GS-SG and 10 mM GSH, i.e. excess reduced GSH; the effect would be that disulfides tend to be reduced, unless they are conformationally protected. However, the other ratio might work as well: 10 mM GS-SG and 1 mM GSH. Now you would expect your Cys13 to be bonded to one of the excess glutathione (mixed disulfed), while the native disulfides remain intact, as they are conformationally stabilized. Of course, you can use a redox system other than glutathione, e.g. cysteine - cystine. Good luck, Hans 2. Did you inhibit S-S bond formation after addition of SDS? Lots of proteins form dimers when unfolded without presence of reducing agent. Adding 20 mM NEM into SDS-PAGE loading buffer is easiest way to prevent this. Assuming that you did this and you actually see intermolecular bond, you can play with reducing agent(s) concentrations. Intramolecular bonds require much higher concentration of reducing agents (e.g., IgG is perfectly happy and not reduced at 1 mM DTT at room temperature). Dima 3. For the first part (i.e. able to selectively reduce the protein at the engineered Cys without breaking the 6 disulfide bonds), this will depend on the solvent accessibility of these disulfide bonds. We have some examples of proteins containing up to 3 disulfide bonds and also free SH groups implicated in the catalysis for which we are able to add up to 2.5 mM of a reducing agent (DTT) without breaking S-S bonds. For the second part, you can use a reversible and specific thiol protecting group, such as MMTS. By doing this you form a S-methylthio-derivative of your protein, which can be easily reduced. Best regards, Rene Wintjens 4. Have you tried selective reduction using cysteine? Cysteine is used to reduce Fab2' to Fab' after pepsin digestions. It tends to reduce surface disulfides and leaves internal disulfides intact. However, you will need to experimentally determine the concentration of cysteine to use. I would try a concentration range of 0.5-20 mM cysteine. Hope this helps. Jeff 5. You could consider to reversibly protect the free cysteine chemically. Treat with sulfite + tetrathionate (ca 10-100 mM) to let the free cysteines react to S-sulfonates and prevent them forming disulfides. Later, when you want the free cysteines, you treet with thiols (ca.1 mM). The problem of regioselectivity for the disulfide cleave, though, remains. Low DTT concentrations or redox buffers with glutathion (GSH/GSSG) could work. Gottfried Original query: We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation), and we also see other higher molecular weight species that are immunoreactive with an antibody to our protein, so maybe we have heterodimerization with other cysteine-containing proteins as well. Can anyone point me to references or protocols that might help us to selectively reduce our protein at the engineered Cys, breaking up the dimers, while preserving the disulfide structure of the native protein? Or might there be a way to reversibly protect the engineered Cys throughout expression and purification to prevent dimerization? Any suggestions are welcome. Thanks! Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
[ccp4bb] structural bioinformatics for dummies
Dear all, Suppose I want to data mine the PDB to find a set of structures containing a motif that is characterized by an unusual conformation of backbone atoms over a short stretch of residues. This structural motif does not correspond to any specific amino acid sequence; the residues could be anything, as long as they conform to the right backbone conformation, within an RMSD of 1 angstrom or so. I then want to filter the list of hits with a solvent accessibility criterion, and then with a sequence criterion at one amino acid position within the motif. This project is similar to one described previously by Richard Jackson and Robert Russell in J Mol Biol (2000) 296, 325-334. They wrote The coordinates of a probe structure are compared to each segment of the same length within representatives from the SCOP database. Secondary structure assignments were taken from the DSSP database, and relative solvent accessibilities calculated using NACCESS. For a person without prior experience in data mining structural databases, where would be a good place to start? Can this type of search be done through a web server? What current software is best suited for the job? Are there any with user-friendly interfaces, helpful tutorials, and the like? Thanks for any suggestions. Evette Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] protein complex crystallisation
What protein concentration are you using in your screen? It sounds like you need to try lower protein concentration, or a screen with lower precipitant concentration, or both. Hampton makes a Crystal Screen Lite with the precipitant concentrations half of what they are in the normal screen (or you can dilute the regular screen). Don't forget that protein concentration is one of the important variables to optimize. For some complexes, I have had best results with protein concentration as low as 2.5-3 mg/mL. Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ron hudson Sent: Saturday, September 20, 2008 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein complex crystallisation Dear CCP4 Community, My apologies for the off topic question to the bb. I am trying to crystallise a small protein-protein complex. I purify as a complex protein after expression in BL21DE3 cells through NI-NTA affinity chromatography. pI of one of the protein is around 10.6 where as another component have 4.0. I am in the screening stage of the crystallisation. During crystallistion process it precipitate very quickly as i add the protein into the crytallisation solution drop. This happens in almost all the condition of the hampton INDEX and crystal screens. I purify this complex in Tris-Cl buffer at pH=8.0. it is happily soluble and donot prcipitate at this pH in the same buffer. I can concetrate this protein upto ~10-15mg/ml. could any one suggest the solution, that will be most appreciatable. Thanks in advance Ron
[ccp4bb] off-topic: selective reduction of surface cysteine
Dear all, We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation), and we also see other higher molecular weight species that are immunoreactive with an antibody to our protein, so maybe we have heterodimerization with other cysteine-containing proteins as well. Can anyone point me to references or protocols that might help us to selectively reduce our protein at the engineered Cys, breaking up the dimers, while preserving the disulfide structure of the native protein? Or might there be a way to reversibly protect the engineered Cys throughout expression and purification to prevent dimerization? Any suggestions are welcome. Thanks! Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Concentrating protein
Another question re: Amicon stirred cells... I also seem to recall seeing 1L size stirred cells in older labs of my youth. My current lab has acquired one of 400 mL, but looking to purchase a bigger one, I can't find any. Any ideas about where we might find one? Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Gina Clayton Sent: Friday, June 27, 2008 7:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Concentrating protein Hi there I quite like the Amicon stirred ultra concentration cell systems. You can put large volumes in, maximum 1 litre size, I think. As well you can attach an inert gas such as Argon or Nitrogen, for the gaseous pressure, this reduces oxidation of your sample while it concentrates. My experience has been that, depending on the filter, the filters are very resistant to various salts even GuHCl, and you get good recovery. I used to concentrate large volumes of protein down to say 50-25ml then switch to the same system, in a much smaller cell i.e. 10ml, to get down to 1-2ml. And they are fairly fast too. I get the impression, perhaps incorrectly, they are not as fashionable as they used to be, but perhaps older labs tend to have them milling about somewhere in the back of a cupboard. So most likely you would only have to buy membranes -PM or YM it think depending on you sample. Hope that helps Gina On Jun 27, 2008, at 9:19 AM, Roger Rowlett wrote: Guenter Fritz wrote: A mild and quick method is to use dry Sephadex G-25. The material will swell and take up all the liquid except molecules larger than ca. 5 kDa. Dear All, we have GCSF protein produced in inclusion bodies. we solubilise it refold it and then concentrate it using proflux system. still the concentration of the protein we get is less and volume is more for us to load in Ion exchange chromatography. is there any simple technique that can be performed in lab without using any hi-fi instrument to concentrate the protein in small volume of buffer. the protein we obtain is about 0.7 mg/ml and we get 450 ml solution. our column is 110ml lab scale and we have to work in that only. i have heard of NH4SO4 precipitation. but it requires protein conc more than 1 mg/ml. kindly help me to progress in my experiment. One of the beauties of ion-exchange chromatography is that it is an excellent concentration step as well as a purification methodology. It may take less time and involve less protein loss to pass all the solution through the IEX column and bind the protein, assuming you have the protein in a low ionic strength buffer at the appropriate pH. Elution in a smaller volume can be accomplished by increasing the NaCl concentration to an appropriate level. In the bad old days before bacterial overexpression, we used to to this routinely to concentrate a liter or more of protein extract to 50-100 mL after elution from a small, high-capacity IEX column. Cheers, -- -- -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] cryoprotectant for crystals in isopropanol
Another anecdote for you: I had crystals that grew in 15% PEG 2000 and 15% isopropanol. Cryos with glycerol melted the crystals. The best cryo I found was with 15% PEG 2000, 15% isopropanol, and 20% PEG 400. It's hard to predict how your crystals will behave with different cryoprotectants-- hopefully you have enough crystals to try several different conditions. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of shivesh kumar Sent: Wednesday, March 19, 2008 3:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryoprotectant for crystals in isopropanol Dear all, The question is regarding the cryoprotectant.We have crystallized a protein in 20% PEG 500 with 10% isopropanol.What should be the cryo we should try for data collection.Also,what percentage of PEG 500 is sufficient enough as a cryo for data collection. We are also trying to crystallize it with higher PEG's also like 1K, 2K, 4K.Lets C.Since the condition is having isopropanol also and it is reported that mounting with isoprop is very difficult and it evaporates as soon as we open the slides. Thanx in advance... Shivesh Kumar
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Thanks to everyone for the great suggestions so far. To clarify and answer a few questions, with the mutated construct we get no protein either secreted or in the pellet. The protein in question is about 20 Kda; with glycosylation at both sites it is around 29 kDa (both in mammalian cells and in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Stephen, We confirmed by PCR that our gene was chromosomally integrated in the many Pichia colonies that we screened for expression, but we did not directly assess for mRNA transcripts. We are indeed using the alpha mating factor secretion signal and have not tested any alternative secretion signals, nor am I aware of any other groups that have tried other signal sequences in Pichia with this family of proteins. We have been extracting proteins from the pellets by a quick alkaline extraction method, variations of which have been published for S. cerevisiae and S. pombe; to judge from our Coomassie stained gels, it is pretty efficient for the Pichia as well. I'd never before thought of checking the homologous residues in the other family members that have been produced in Pichia. The residues at these positions by sequence alignment are D, P, G, and S. However, when I do a structure-based alignment with the one other family member that has been crystallized, I see that structural conservation at these sites is poor; they are on loops that look completely dissimilar between the proteins, so I don't think I can read much into the substitutions. Thanks for your help. Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -Original Message- From: Stephen Weeks [mailto:[EMAIL PROTECTED] Sent: Wednesday, March 05, 2008 10:43 AM To: CCP4BB@JISCMAIL.AC.UK; Radisky, Evette S., Ph.D. Subject: Re: Removal of glycosylation sites in Picha expression construct Evette, This is quite intriguing. At the outset I want to say that just because glycoslyation is not essential for function it still may be absolutely necessary for correct trafficking. Obviously in your case the mutant has been made successfully in CHO cells, assuming the mutant transcript is present in Pichia I wonder if this is an effect of fusing it to the alpha mating factor secretion signal. Have you - or anyone else for that matter - tried alternative secretion signals ? Sorry but I have two more questions for you. How do you lyse you cells to see production of the protein in the pellet ? I found boiling Pichia in SDS buffer pretty inefficient, do you use glass beads ? Secondly I'm curious as to what residues are present when you align you protein to the related non-natively glycosylated proteins (that where successfully expressed in Pichia) specifically at the site of glycosylation. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452
[ccp4bb] Removal of glycosylation sites in Picha expression construct
Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Question regards to binding affinity of the protein complex?
I do not know about the affinity required for gel filtration, but I have had success crystallizing a number of protease-inhibitor complexes just by incubating and setting up drops with equimolar amounts of the two components. It is probably to our advantage that we have functional inhibition assays that allow us to be very precise in achieving the equimolar ratio. This has worked for a lot of complexes with Kd in the low nanomolar to low picomolar range, but we recently also had success with a complex where Kd was 14 micromolar. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ngo Duc Tri Sent: Wednesday, September 19, 2007 6:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question regards to binding affinity of the protein complex? Dear CCP4 Users, I'd like to solve the structure of the protein-protein complex. I intend to purify and incubate the complex then run gel-filtration before setting crystal. I'd like to know your experience about the Kd value of the interaction in order to get the complex after running the gel-filtration. In other words, what is the lowest Kd that we can purify the protein complex using gel-filtration? And if the binding affinity is too low to purify the complex, is it possible to get the crystal of the complex if I just incubate and set crystal of this mixture? Thank you very much for all of your advice! My best regards, TriNgo Sungkyunkwan University
[ccp4bb] Twin refinement with SHELXL
Dear All, I have 1.4 A data and a molecular replacement solution for a crystal indexed as C2, with beta approximately equal to 90. Refinement with refmac is progressing poorly, and intensity statistics (Truncate) and other twinning tests (xtriage) suggest pseudo-merohedral twinning with a twin fraction around 0.44. I want to try refining in SHELXL with a twin-specific target function, but I have some questions about how to get started. SHELX documentation indicates that the reflection file should contain intensities rather than amplitudes, and preferably unmerged data. I have been refining against an mtz file that was integrated with MOSFLM, scaled and merged with SCALA, converted to amplitudes with TRUNCATE, then had freeR flags assigned. I am getting the mtz2various patch recently mentioned on the bb, that will make an mtz file into a proper SHELX hkl file, but which mtz file do I convert? The mtz output by TRUNCATE contains both F's and I's, so I could use that one to get measured intensities, or do I actually need to go back further to get my unmerged reflections? A second question relates to Rfree flags - should I transfer the ones I've been using up until now? They were a randomly assigned 5%. Now I've read that using the thin shell method is better for twinned crystals and NCS, but I don't know exactly how that is done... any recommendations? Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-2857 (lab)
Re: [ccp4bb] Multiple nucleation
In addition to trying lower concentrations of PEG and protein, you might consider whether aggregation in your protein stock or vibration in your crystallization environment are contributing to the problem, and how they might be minimized. It might be obvious, but we have found that it is important for protein stocks to be filtered or centrifuged to remove aggregates immediately prior to setting up drops, and if the protein solutions have been sitting around for a few hours or a few days, they must be centrifuged again. We also had a stupid incident recently where conditions that had previously generated high quality crystals were suddenly producing thousands of tiny crystals instead, and the problem was traced to a clinical centrifuge that had been moved to an adjoining bench and was vibrating the crystallization shelf. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-2857 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jobichen Chacko Sent: Thursday, March 29, 2007 7:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Multiple nucleation Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi
