[ccp4bb] OFF TOPIC - zebra printer labels for SBS format plates
Hello all, Sorry for the off topic post. I am looking for alternate (and more reasonably priced) sources for label rolls for our Zebra printer for labeling crystallization experiments. We use Formulatrix rock imagers and rock maker software but am interested in hearing from anyone that uses Zebra printers for labeling SBS format plates about your source and approximate cost/roll. Please respond to me privately and I will post a follow-up summary. U.S. sources would be most appropriate for my purposes but I am happy to collect sources from elsewhere in the world to compile in my summary. thanks in advance for your suggestions/advice. sincerely, Eric Eric Larson Structural Research Boehringer Ingelheim Pharmaceuticals Ridgefield, CT USA
[ccp4bb] open Structural Biologist position at Rheos Medicines
Hello Bulletin Board members. We are seeking to expand our structural biology group at Rheos Medicines in Cambridge, MA. Please see the details here: https://rheosrx.com/careers/job-detail/?job=364979 and feel free to reach out to me if you are interested or if you know someone that may be. thank you and stay safe. Eric Larson eric.xt...@gmail.com To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Change cell parameter
Hi Zhiyi, This is very easily done using Pointless. From the gui, click "Match index to reference", enter your two mtz files and run. ERic Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Wed, 8 Jun 2011, Vellieux Frederic wrote: Zhiyi Wei wrote: Dear all, I have a P2 derivative dataset with beta=89.6. I try to change the beta to 90.4 to be consistent with the native dataset. Should I do sth with the HKL, like applying a matrix? Thanks a million! Best, Zhiyi Hi, Personally I would use sftools (no ccp4i GUI), to be run in a terminal sftools read mymtz.mtz set cell [then you specify the new cell] write mynewmtz.mtz stop However, before changing cell parameters I would think twice... Further, each data set in an mtz file can have its own cell dimensions. Differences in unit cell parameters of less that 1% (I think this is the consensus) are still isomorphous, over this you have non-isomorphism. There is a paper on this (Crick ?). Fred.
Re: [ccp4bb] Convert .cif to mtz
Hi Madhu, D*trek is what you processed your data with. In the ccp4i gui, select the program "Dtrek2mtz" and then in the field that says "convert scaled data output from ... ", select "d*trek" from the pull down menu. Go to http://www.ccp4.ac.uk/html/dtrek2mtz.html for more information. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Fri, 24 Jun 2011, Madhu shankar Madhu shankar wrote: HI, I indexed and integrated data from RAXIS(Rigaku) detector on 'Crystalclear'. It gave me a .cif file as output.I am not able to read this file in ccp4 to carry out rest of the analysis in ccp4. I want to know how to convert this cif file to MTZ. Thanks Madhu
Re: [ccp4bb] Convert .cif to mtz
Madhu, I just noticed that your message said you "indexed and integrated" your data. Did you scale it with d*trek too (that needs to be done before using Dtrek2mtz) or are you looking to use the integrated d*trek reflections for subsequent scaling in ccp4 with scala? Eric On Thu, 23 Jun 2011, Eric Larson wrote: Hi Madhu, D*trek is what you processed your data with. In the ccp4i gui, select the program "Dtrek2mtz" and then in the field that says "convert scaled data output from ... ", select "d*trek" from the pull down menu. Go to http://www.ccp4.ac.uk/html/dtrek2mtz.html for more information. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Fri, 24 Jun 2011, Madhu shankar Madhu shankar wrote: HI, I indexed and integrated data from RAXIS(Rigaku) detector on 'Crystalclear'. It gave me a .cif file as output.I am not able to read this file in ccp4 to carry out rest of the analysis in ccp4. I want to know how to convert this cif file to MTZ. Thanks Madhu
Re: [ccp4bb] Convert .cif to mtz
Hi Madhu, You don't need to use scala, d*trek can do the job as well. The easiest thing to do is to continue with d*trek to scale and merge your data and then input this scaled and merged file into Dtrek2mtz. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Fri, 24 Jun 2011, Madhu shankar Madhu shankar wrote: HI Eric, I havent yet done the scala ,i have just indexed and integrated the data in d*trek. I tried converting the .cif in Dtrek2mtz but thats failed. Thanks Madhu
Re: [ccp4bb] Off topic - transformation problems
On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
Re: [ccp4bb] Off topic - transformation problems
oops - I just caught that the empty pET20 did transform well so the other suggestions about toxicity are probably more accurate. On Thu, 14 Jul 2011, Eric Larson wrote: On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
Re: [ccp4bb] Off Topic: How to delete loops from a protein
Hi Obayed, you could give in situ protolysis a try. This is where you add a bit of protease along with you target protein to the crystallization drop. It has been quite successful for the folks at the SGC. Here are the relevant references: Dong A, et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) Wernimont A, Edwards A. In situ proteolysis to generate crystals for structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 18 Jul 2011, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Concentrating a protein solution - subbu
Hi Subbu, You got crystals at 1mg/ml so you probably don't need to concentrate your protein any higher, especially since you suggest that concentrating beyond that is problematic. Instead, you may want to try to optimize the crystallization condition you have already identified. Some possible things to try: additive screen, include specific ligands, different temperature, different ratios of protein solution to crystallization solution, seeding, different crystallization methods (hanging vs. sitting drop, batch, diffusion,...), ... good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote: Dear All: We have been trying to crystallize a protein which is large - > 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
Re: [ccp4bb] Concentrating a protein solution - subbu
See below. I believe Ray intended this to be sent to the entire bb and especially to Subbu! And in reading Ray's message, I am reminded that I forgot to mention what he may be hinting at. You should also try to optimize by creating a fine grid screen around your identified condition varying in particular pH and precipitant concentration. best, Eric On Thu, 21 Jul 2011, ray-br...@att.net wrote: >What is the pI? Perhaps the protein will be more soluble close to this pH? >You could increase the salt and/or add some glycerol or a >cofactor? > >Normally crystallization is most affected by the pH, 2 - 10% glycerol or >temperature 4 - 25 degrees C. >Cheers > >Ray Brown > >_________ >From: Eric Larson >To: CCP4BB@JISCMAIL.AC.UK >Sent: Thu, July 21, 2011 1:40:51 PM >Subject: Re: [ccp4bb] Concentrating a protein solution - subbu > >Hi Subbu, > >You got crystals at 1mg/ml so you probably don't need to concentrate your >protein any higher, especially since you suggest that >concentrating beyond that is problematic. Instead, you may want to try to >optimize the crystallization condition you have already >identified. Some possible things to try: additive screen, include specific >ligands, different temperature, different ratios of >protein solution to crystallization solution, seeding, different >crystallization methods (hanging vs. sitting drop, batch, >diffusion,...), ... > >good luck, > >Eric > > >Eric T. Larson, PhD >Biomolecular Structure Center >Department of Biochemistry >Box 357742 >University of Washington >Seattle, WA 98195 > >email: larso...@u.washington.edu > > >On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote: > >> Dear All: >> We have been trying to crystallize a protein which is large - > 100 kDa. >> This is soluble but the best we can get is about 1 mg/mL. >> It did crystallize but did not diffract well. Efforts to increase the >> concentration has been unsuccessful. I am wondering whether >there are methods that others use to increase the concentration other that >using amicon columns. >> Any help will be appreciated. >> Thanks >> Subbu >> > >
Re: [ccp4bb] pdb_extact and refmac5.6
Hi Jan, Have you tried using the x.refmac file that is created instead of the log file. It is in cif format and may be in the "DepositFiles" directory. Hope you are well and talk to you later, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Wed, 27 Jul 2011, Jan Abendroth wrote: Hi all, we typically use pdb_extract to generate pdb files with a good header. There seems to be an issue with logfiles from refmac5.6.0117, distributed with the current ccp4 version, and pdb_extract. pdb_extract version-3.006 is distributed with ccp4-6.2.0. While reading the refmac log file, pdb_extract chokes with a segmentation fault. When run without reading in the refmac log file, the program complains about the following missing link, both in the linux and osx distribution of ccp4. Can not open file (/usr/local/ccp4/ccp4-6.2.0/ccp4-6.2.0//src/harvest_app_/pdb_extract/pdb-extract-data/mmcif_pdbx_2.items) in get_lines_from_file pdb_extract version-3.010, as downloaded form the rcsb webpage also chokes on the refmac5 logfile with a segmentation fault. Any ideas how to treat the new refmac log files in pdb_extract? Thanks a lot. Jan -- Jan Abendroth Emerald Biostructures Seattle / Bainbridge Island, WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com
Re: [ccp4bb] Freezing crystal
Hi Theresa, A good place to start when searching for suitable cryo conditions are the tables in these references: Garman, et al. J. Appl. Cryst. (1996). 29, 584-587. McFerrin, et al. J. Appl. Cryst. (2002). 35, 538-545. hope they help and good luck. Eric __ Eric Larson Boehringer Ingelheim Ridgefield, CT Date:Sun, 5 Feb 2012 22:49:25 + > From:"Theresa H. Hsu" > Subject: Freezing crystal > > Hi all > > Is there a list of conditions to be tried *first* for cryoprotectant? My > crystals diffract at room temperature capillary but no in 30% PEG 400. > Crystals are from 2 M ammonium sulfate. > > Thank you. > > Theresa > >
Re: [ccp4bb] How to align a sequence to a know profile
3-d coffee and expresso will align sequences without structures with sequences that do have structures in a structure-based sequence alignment: http://www.tcoffee.org/Projects_home_page/expresso_home_page.html perhaps this is along the lines of what Yuan is looking for? __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 1 Jun 2010, Thomas Juettemann wrote: Does chainsaw not the opposite? Pruning a coordinate file based on non-conserved residues identified in a MSA? Yuan has a MSA of known structures and a sequence he wants to add to it. I am always keen to learn about alignment programs, it would be great to know how to use chainsaw for this problem. On Tue, Jun 1, 2010 at 02:20, Eleanor Dodson wrote: chainsaw does just that eleanor 商元 wrote: Hello, everyone, I've a protein sequence of known domain. Based on structure alignment, I've got a alignment of those with known structures. Then how to add my sequence to the alignment?Any suggestions? Regards, Yuan SHANG
Re: [ccp4bb] Off-topic: hollow protein crystals
(* which still shows "To be published", 3 years after we published it - does the PDB not figure this out automatically?) The authors need to take the initiative and let the PDB know when their structures have been published. The correspondence from the PDB people when they are curating the structure deposition says something along these lines: "When the primary citation associated with your entry is published, please notify us at depo...@deposit.rcsb.org and provide pubmed ID (if available), journal name, volume, page numbers, title and authors list." ______ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Wed, 2 Jun 2010, Nat Echols wrote: On Wed, Jun 2, 2010 at 12:07 PM, Andre Ambrosio wrote: We have recently obtained crystals from a small protein, and interestingly, at least for me, they are hollow trigonal rods (please see pictures attached). Just out of curiosity, has anybody ever seen such feature for protein crystals before? Yes, I had very similar crystals once (PDB ID 2i6f*). They were in the I4 space group, and the lattice formed two solvent channels, one large, one small, which I assumed ran the entire length of the crystal. The chains adjacent to the large solvent channel were poorly ordered and nearly uninterpretable in some datasets, so my best guess is that the hollow crystals were the result of this disorder. Fortunately, it didn't appear to have any effect on the diffraction quality. -Nat (* which still shows "To be published", 3 years after we published it - does the PDB not figure this out automatically?)
Re: [ccp4bb] process data in R32
Hi Ray, In HKL2000, by default (i.e. - without changing anything manually) for R32/H32, Denzo/Scalepack leads to a .sca file that has hexagonal unit cell parameters in the header but has the space group listed as R32. Scalepack2mtz in ccp4 does not like this mismatch so you have to simply change the space group name from R32 to H32 in the space group box in the "Extra Information for MTZ File" section of the scalepack2mtz gui and all should then be fine. good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Wed, 11 Aug 2010, xaravich ivan wrote: Hello Ray, You can contact the author of this paper. It seems they used denzo scalpack for R32. Ivan On Wed, Aug 11, 2010 at 8:53 AM, Eleanor Dodson wrote: If you cant import a file with symmetry labelled R32 but with a=b and alpha=beta = 90, and gamma = 120 then something is wrong - the symmetry checking looks at both name and cell.. or at least it used to.. The output mtz would have symmetry name changed to H32, but that is just a name - in each case the sym ops and cell should be h32 style.. Eleanor Ray Brown wrote: Hi, I have read all of the past messages about the confusion of H32 and R32 and I want to get the correct indexing sp that I can import the .sca file into CCP4.. I use DENZO and SCALEPACK in HKL2000. If you read the manual then it states that space group R32 should only be autoindexed by DENZO on a primitive lattice using the DENZO keyword H32. This indeed gives a primitive unit cell and not the hexagonal setting apparently demanded by CCP4 programs. What do you do next? It is possible in SCALEPACK to reindex the H32 primitive data into a rhombohedral hexagonal setting which SCALEPACK calls R32. Or course you cannot import this .sca file into CCP4. Why not? I can go into the .sca file and edit the header and change the R32 label to H32. Does this screw up the indexing? If anybody has successfully used DENZO/SCALEPACK/CCP4 in space group R32 then please tell me how to do it correctly. Cheers. Ray email ray-br...@att.net
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
Hello Qiangmin, Here are links to a few web resources that have tips for membrane protein crystallization that may help with your optimization strategy: http://kb.psi-structuralgenomics.org/update/2009/06/full/th_psisgkb.2009.25.html http://web.emeraldbiosystems.com/blog/bid/33916/8-Practical-Tips-for-Membrane-Protein-Crystallization http://mcl1.ncifcrf.gov/nihxray/Tips-and-Tricks_Crystallization_Membrane_Protein.pdf good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Mon, 30 Aug 2010, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] Deposition of riding H: R-factor is overrated
Hi Mark,
I assume you deposited the mtz? This is what Ethan was referring to - the pdb
does not do well with maintaining all the relevant columns when submitting the
mtz file. However, if you convert your mtz to cif yourself and make sure it
has all the columns you would like to include and then submit this cif file to
the pdb, all the information is retained.
Eric
__
Eric Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195
On Thu, 16 Sep 2010, Dr. Mark Mayer wrote:
Ethan wrote
I believe that deposition of Fc Phic FOM should be required.
Certainly it should be the recommended practice.
For the same series of structures I just deposited, which started the the
riding H discussion, my mtz file had Fc Phic FOM + other data put out by
Phenix - pavel can elaborate. rcsb stripped almost all of this and the
processed file has only:
HKL, Flag, Fc, SigmaF and FOC :{
What's a structural biologist to do?
--
Mark
Re: [ccp4bb] **Possible spam**cryoprotetant for 35% Dioxane
Hi Jerry, A great reference for initial cryocondition searches for many "standard" crystallization solutions is the tables in: J. Appl. Cryst. (2002). 35, 538-545 [ doi:10.1107/S0021889802009238 ] The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images M. B. McFerrin and E. H. Snell On the bottom of page 542 it says for 35% dioxane as the precipitant in Hampton Crystal Screen II condition # 4: 25% glycerol, 25% PEG 400, 20% ethylene glycol, 15% propylene glycol (1,2-propanediol) good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Wed, 20 Oct 2010, Jerry McCully wrote: | Dear All; | | We just got some crystals from 35% (v/v) Dioxane. We are going to collect some data soon. | | Does anyone have the experience with the cryoprotectant in this condition? | | Thanks a lot, | | Jerry McCully | | | |
Re: [ccp4bb] how to optimize small rod-shaped crystals
Hi Yibin, I cannot tell the actual scale of your crystals but judging by the curve of the drop outline, these crystals look to be plenty big enough to mount in a loop to stick in the beam and test diffraction. If indeed they are too small to mount a single crystal, and you are mainly interested in testing to see if they are salt, you can gather up several of them in your loop and shoot the group - this won't be useful for data collection if it turns out to be protein but will be sufficient to tell you if your crystals are salt. good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 16 Nov 2010, yybbll wrote: | Hi, everybody, | | I try to crystallize one membrane protein. All crystals were grown by handing-drop vapor diffusion at 20 degree. A protein solution containing | about 8-10mg/ml protein in 20mM Tris (pH7.5), 0.017% DDM, 100mM NaCl, 10% glycerol, 2mM DDT was mixed with an equal volume of a reservoir solution | containing 45% PEG200, 0.1 M phosphate/citrate (pH4.2). First crystal appeared in the drop within 4 days. And one week a lot of crystals appeared in the | drops. | | Our question is all of these crystals are too small to check them by X-ray diffraction and SDS-PAGE. We are not sure they are protein crystals or salt | crystals. Our condition seems difficult to produce salt crystal. But I am a little warry because we use reloaded our sample to small Ni-resin column to | reduce the concentration of detergent. Maybe some nickel ion dropped off, and then our protein sample contained some this ion. And nickel ion may react | with phosphate, and then produced nickel phosphate crystal. Could somebody tell me if it is possible? | | I attach some photos of our crystals. Could somebody give me some suggestions about how to optimize this type crystal to get bigger crystal? | | Thanks a lot! | | Yibin | | | 2010-11-16 | | _ | yybbll | | _ | 发件人： Laurie Betts | 发送时间： 2010-11-16 17:13:32 | 收件人： CCP4BB | 抄送： | 主题： [ccp4bb] expression of Cys-rich small protein | All - | | We are trying to express for structural studies a 257 AA eukaryotic intracellular (also possibly nuclear) protein (predicted to be single domain | all-helical) that has 12 Cysteines. No known metal-binding function not that it couldn't happen. So far (E. coli) it expressed solubly as MBP fusion | (with an N-terminal region deleted predicted disordered) until cleavage of MBP, then it's not soluble, including detergents added. THe MBP fusion is | usually soluble aggregate so we assume that our part is not folded right. We have so far assumed it needs a lot of reducing agent (5 mM DTT or TCEP). | Thinking of trying chaperones and insect cells next. | | Any experience out there that might help? Mostly I wonder about all the cysteines. Don't really know if that is the problem. | | Laurie Betts | |
Re: [ccp4bb] Off Topic - Nickel Column
Hello Dan, another denaturing option worth trying is to do it on the nickel column. Purify via your normal means to start but then while your protein of interest is bound to the nickel resin (with the sticky partner bound to it), you can wash with buffer containing increasing amounts of urea (or Gd-HCl) and then continue to wash while slowly decreasing the amount of denaturant. Finish off with a very thorough wash in your original denaturant-free wash buffer and then elute. Your protein of interest should remain bound to the Ni column throughout but the sticky protein should be washed away and (if you are lucky) your protein will then refold while bound to the column and you will elute native protein minus your troublemaker. You may not even have to go all the way up to 8M urea or 6M GdHCl, only using a small amount of denaturant may be sufficient to remove the troublemaker and your protein of interest may not be completely denatured in the process making refolding less of a proble! m. good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Wed, 17 Nov 2010, Michael Thompson wrote: | Dan, | | You could try a denaturing purification to get rid of the binding partner. | | First, take your cell lysate and spin it down at high speed to remove insoluble contaminants. (You probably do this already.) Then take your clarified lysate and dialyze it into buffer containing 6M Guanidine HCl. This will unfold everything, including proteases (no proteolysis under these conditions), and break the interaction between your protein and its binding partner. Then you can spin again at high speed to remove any additional aggregates and load this denatured sample onto the nickel column. The His-tag will still stick if the protein is unfolded in the presence of 6M GdHCl. Elute the protein in the same buffer with 6M GdHCl plus high imidazole concentration. Then you can take your eluent and dialyze out the GdHCl to refold the protein. | | Refolding (in the test tube, anyway) is not possible (practical?) for all proteins, but it often works very well. | | Good luck, | | Mike Thompson | | | | | - Original Message - | From: "Daniel Bonsor" | To: CCP4BB@JISCMAIL.AC.UK | Sent: Wednesday, November 17, 2010 8:49:37 AM GMT -08:00 US/Canada Pacific | Subject: [ccp4bb] Off Topic - Nickel Column | | I have a His-tagged protein which I am coexpressing with it's binding partner to prevent proteolysis. Once on the Nickel column I can remove 80% of the partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight. However the last 20% is difficult to remove, even if I reload the Nickel column and flush a further 2l of salt solution. I am wondering if I can increase the pH to 9.0 or 9.5. It should not effect the binding of His for the Nickel as the His-tag has to be deprotonated to bind, though will it causing stripping of the Nickel? | | Thanks | | | Dan | | -- | Michael C. Thompson | | Graduate Student | | Biochemistry & Molecular Biology Division | | Department of Chemistry & Biochemistry | | University of California, Los Angeles | | mi...@chem.ucla.edu |
Re: [ccp4bb] Problem with finding of spots
Hi Petr, You don't have to rely on automatically picked spots if you do not trust them. Have you tried manually selecting spots and then limiting the high resolution cutoff for indexing? Oftentimes that is sufficient to get past at least the indexing step. hope that helps, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 23 Nov 2010, Petr Kolenko wrote: | Dear colleagues, | | I am working on one dataset that is hard to process. The data are about 3A of resolution. As we are not able to reproduce the experiment again, I have to | use this one, collected in a dirty way. | The problem starts immediately with finding of spots. I have tried HKL2000, XDS, D*trek, ipmosflm, imosflm, but none of them gave a good read-out of the | images. All the programs find some spots in wrong positions and the real spots are not covered. Here is an example: | | http://kolda.webz.cz/image-predictions.jpg | | The data were collected in-house, Saturn 944++ CCD, and all the necessary information should be in the header properly. I checked the distance, other | parameters, but the problem is with finding of "correct" or "real spots" on the image. This should be even header-independent, should not? All the | programs fail (or even crash) in this routine. Does anyone have any suggestion, please? | | Btw, we have several structures in the PDB from this experimental setup. This is the first problem I have met. | | Many thanks for any response. | | Petr | | -- | Petr Kolenko | petr.kole...@biochemtech.uni-halle.de | http://kolda.webz.cz | |
Re: [ccp4bb] Structure based and motif based sequence alignment
Hello Bashir, If I understand your question correctly, Expresso and 3D-Coffee from the T-coffee people will do just what you want. They align sequences while utilizing any available structural information. http://www.tcoffee.org/Projects_home_page/expresso_home_page.html hope that is what you are looking for, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Fri, 26 Nov 2010, Muhammed bashir Khan wrote: | Dear All; | | I have structures of two protein one full-length while the other truncated | at the c-terminus(one from prokaryote while the other from eukaryotes). | Now I want to do the sequence alignment of these two proteins from all | species in such a way that the structure based sequence remain constant | while extending the sequence only at the c-terminus. Remember the | structure are known only for the two proteins. | | Any suggestion will be highly appreciated! | | Regards and have a nice weekend. | | Bashir |
Re: [ccp4bb] Saxs reviews or books
Hello Rojan, This paper from last year may be a good place to start. It provides a nice "road map" through the small angle scattering experiment that a non-expert can follow while also describing the scattering data so that a non-expert can more critically evaluate it. I found it very informative. David A. Jacques and Jill Trewhella. Small-angle scattering for structural biology—Expanding the frontier while avoiding the pitfalls. Protein Science Volume 19, Issue 4, pages 642–657, April 2010. DOI: 10.1002/pro.351 http://onlinelibrary.wiley.com/doi/10.1002/pro.351/abstract hope that helps, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Tue, 11 Jan 2011, Rojan Shrestha wrote: Hello: I am very novice about Small Angle X-ray Scattering. I am looking for introductory books or review papers. Could you recommend this type of document? Regards, Rojan
Re: [ccp4bb] So many clashes
Hi Careina, Are you using riding hydrogens during refinement? The default in Refmac is to use hydrogens only if present in the input file - change this setting under the "Refinement Parameters" to "generate all hydrogens". This significantly helps with clashes. Also, did you check the quality of your molecular replacement input model? If this model had a lot of clashes, then you are starting with the same problems and it can be difficult to overcome. At better than 2 ang. resolution, one of the automated building programs (arp/warp, resolve, buccaneer, ...) should be able to rebuild much of your model and may help with your clash problem too. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Tue, 18 Jan 2011, Careina Edgooms wrote: Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well to electron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
Re: [ccp4bb] buffers for crystallization
Hi Chandan, What do you mean by problem in crystallization? Too much precipitate, too many clear drops, ...? If you state the specific problems you are experiencing it is easier to make specific suggestions. By 388 buffers, do you mean that you have only tried 388 crystallization conditions or ~4 commercial screens? That really is not a lot of conditions to try these days. Have you tried with and without Zinc? Are there other known ligands for your protein you can try to use? Have you experimented with other variables such as protein concentration, temperature, ratio of protein solution to reservoir solution, volume of drop, crystallization method (hanging drop vs sitting drop vs. batch vs. ...), etc. Perhaps the buffer your protein is in going into the crystallization experiments needs to be optimized. Perhaps it is your protein itself that is problem. Do you have a cleavable affinity tag for purification? Try crystallizing both the cleaved and non-cleaved protein. You may need to design other constructs of your protein. Move the tag to N- or C-terminus. Make a series of truncation mutants. Maybe you have disordered domains that are preventing crystallization - remove them. Are there homologous proteins in other species you could try? And the list goes on - there is a near limitless number of things that can be tried. hope that will at least get you started. good luck. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 27 Jan 2011, chandan kishore wrote: Hi Everyone, I have to crystallize one protein which contains a zinc fingure motifs, I am facing a problem in crystallization. I already used 388 buffers . Can anyone suggest me some books or papers freely available online on Buffers required for crystallization. Thanks
Re: [ccp4bb] How to align electron density maps?
Hello Hui, This is also done quite easily in coot. Go to Extensions --> maps --> Transform map by LSQ model fit... Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Fri, 28 Jan 2011, RONG hui Rong wrote: It does work well. That is great! Many Thanks! On 1/28/11, Pavel Afonine wrote: Hi Hui, (sorry for non-ccp4 recipe) IF I understand correctly what you want to do, then I guess you can achieve this using Map Superposition: http://www.phenix-online.org/documentation/superpose_maps.htm and/or Structure Comparison tools in PHENIX: http://www.phenix-online.org/documentation/structure_comparison.htm where you can superpose many models and corresponding maps, and see all them in one display, along with highlighted differences. Nat Echols, the author of both tools, is someone to ask should you have any questions. Pavel. On Fri, Jan 28, 2011 at 10:15 AM, RONG hui Rong wrote: Dear All, I have the problem as follows, but I can not find the corresponding solution. Can somebody give me some suggestions? Many Thanks, Hui Rong * [PyMOL] aligning electron density maps Eksterowicz, John Wed, 03 Mar 2004 15:03:43 -0800 I have a pdb and xplor file which contain two units of the same protein/inhibitor complex. What I am trying to do is align the two proteins to each other so I can compare the subunits. I can accomplish this by reading in the pdb file twice and aligning the b unit from the second pdb file to the a unit of the first pdb file. I then split everything into objects then delete what i don't want. In the end I have subunits a and b aligned to each other. I would like to be able to do the same thing for the electron denisty map. Is there a way to align maps? If I read in a protein and a map, then align the protein to another protein, the protein moves, but the map remains in it's original location. Can the map coordinates be transformed somehow? Is it possible to create some association with the protein and the map such that when the protein is aligned, the map will follow? Thanks, John * On 1/28/11, RONG hui Rong wrote:
Re: [ccp4bb] off-topic: tag removal
Hi Phil, Must you tag your protein? Have you tried to express it without a tag? Perhaps you can purify the un-tagged protein with a series of chromatographic (or other) steps - particularly if it expresses well. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Wed, 23 Feb 2011, Philipp Ellinger wrote: Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] Is there any program for specifically calculating Rvalue in CCP4
Dear TingWei, Can you provide more details of what you are trying to do and why, and how you manipulated the model for which you want to determine the R-values? Is it data you collected and a model that you have been working on or is it a structure and its data from the PDB that you are working with or perhaps something else? What do you mean by refined yourself without help from any program? What did you do? Manually move some atoms by changing the coordinates in the PDB file without even the aid of a graphics program such as Coot or O or Xtalview? Did you use the data (an electron density map) to aid with your manipulation/"refinement"? How? If you just want R-values without doing any refinement, you can plug your manipulated model and dataset into Refmac and do zero refinement cycles, which will give you some relavant statistics. A non-ccp4 option to get statistics for particular models and datasets is phenix.model_vs_data in the Phenix package (http://scripts.iucr.org/cgi-bin/paper?he5476) hope that helps, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 3 Mar 2011, Ting-Wei Jiang wrote: Dear all experts, I'm trying to calculate R-value (and free R) specifically which is between data and the modified structure(refined by myself without help from any program).I've looked the program for calculating a long while.Actually,I found one named Rsearch (CCP4 supported).Nevertheless,I cant find Rsearch in CCP4 package. Could anyone direct me on how to get this value? thanks in advance. :> TingWei
Re: [ccp4bb] Question about movie making
Hi Mark (and Matthias), I'm not sure if "Windows Movie Maker" is the same as (or maybe the predecessor to?) ""Windows Live Movie Maker" (http://explore.live.com/windows-live-movie-maker?os=other), but this is what I used recently to string together a series of png images from pymol to make a wmv movie of a protein motion. With Windows 7, the movie maker is no longer included by default with the OS so it must be downloaded (for free) and installed. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 7 Mar 2011, Matthias Haffke wrote: Hi Mark, just use the "Windows Movie Maker" - it's easy to use and comes along with Windows (or can be downloaded via microsoft.com). This will help you to generate .wmv output files of any kind of video and / or picture files you provide as input files. The best possible quality output format in Windows Movie Maker is 1080p, which should give you a good quality. If there is still the need to convert it to .mpeg, you can use ASF Converter, which is freeware as well (see here: http://www.boilsoft.com/download.html ). Matthias _ Date: Mon, 7 Mar 2011 16:21:02 -0500 From: mjvdwo...@netscape.net Subject: [ccp4bb] Question about movie making To: CCP4BB@JISCMAIL.AC.UK All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to "stitch together" the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University
Re: [ccp4bb] Met auxotroph and aa-mix
Hi Eric, If you are trying to express your Se-Met protein, it is typically done in a defined media starting with a minimal media base so a mixture of amino acids, particularly those that are essential, is needed. You likely will not get very high yields of SeMet-labeled protein if you simply add SeMet to a rich media like LB or TB, etc. for expression. The B834 auxotroph is not necessary, however. We typically use standard BL21(DE3) cells and follow the procedure by Studier FW. Protein Expr Purif. 2005 May;41(1):207-34 (http://www.ncbi.nlm.nih.gov/pubmed/15915565) to get high yields of SeMet protein. Still requires a defined media though. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Sat, 30 Apr 2011, wrote: Dear all, I want to make a Se-Met-labeled protein using the Met auxotroph strain B834. All the protocols I've found require addition of aa-mix. Has anyone expressed in this strain without the aa-mix? Any ideas and suggestions? Thanks in advance! Eric
Re: [ccp4bb] Pymol questions
Hi Eric, On Wed, 4 May 2011, jlliu liu wrote: Hi All, I have two questions for Pymol. the pymol wiki is your friend (http://www.pymolwiki.org/index.php/Main_Page) as is the pymol users mailing list (http://sourceforge.net/mail/?group_id=4546). Follow the link to subscribe. 1. Can you write out the PDB file after structural alignment? yes you can (http://www.pymolwiki.org/index.php/Save) 2. How to only show structure that is several angstrom from the ligand? use the selection algebra rules (http://www.pymolwiki.org/index.php/Selection_Algebra) happy pymoling, Eric Thanks a lot in advance! Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
Re: [ccp4bb] Low resolution refinement
Dear Joane, We have had very good luck refining low resolution structures with dramatic improvement using several newish options in refmac - particularly when NCS is present! Those options are jelly body restraints, automatic NCS restraints, and map sharpening. Here is a description cut-and-pasted from Garib's presentation (www.ccp4.ac.uk/schools/China-2011/tutorials/refmac_tutorial.pdf) V) Low resolution refinement If you have older version of ccp4 then you can follow the instructions described in the presentation: www.ysbl.york.ac.uk/refmac/Presentations/Refmac_Erice_workshop.ppt slides 45-50 Full description of new features are in: www.ysbl.york.ac.uk/refmac/data/refmac_news.html In the new version of ccp4 there are options for jelly body, automatic NCS and map sharpening. a) Jelly body is under “Refinement Parameters”. You need to click “Use jelly- body refinement with sigma”. Change sigma to 0.01 or 0.02. This value defines “jelliness”. Smaller value means tighter restraints b) Automatic NCS restraints are under “Setup Non-Crystallographic Symmetry”. You need to cick “use automatically generated local NCS restraints”. You can also use global NCS c) Map sharpening is under “Monitoring and Output Options”. You need to click “Perform map sharpening with B value 20.0”. B value should a little bit smaller than Wilson’s B value. more in depth information can be found here: www.ccp4.ac.uk/schools/China-2011/talks/refmac_Shanghai.pdf This is definitely something you should explore. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 19 May 2011, David Schuller wrote: > This reminds me of: > > Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: Sequence > analysis and crystal structure determination > Barycki JJ, O'Brien LK, Birktoft JJ, Strauss AW, Banaszak LJ > Protein Science (Oct 1999) Vol 8, pp 2010-2018. > > In which the protein in question also had one monomer forming a dimer about a > crystallographic axis, and two monomers forming a dimer elsewhere in the > asymmetric unit. > > A portion of the molecule had messy density, which caused difficulties > literally for decades. The eventual solution was to switch to a different > species (human). After which, a MR solution of the original porcine data was > possible. I believe the disorder was judged to be intrinsic. > > > > On 05/19/11 09:02, Joane Kathelen Rustiguel wrote: >> Dear all >> >> >> I am refining a structure at 3.4 A resolution that contains 3 molecules in >> the >> a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric >> functional structure expected for this class of proteins. The other two >> chains >> B and C, which also form the functional dimer, seem to be, somehow, a lot >> more >> flexible than chain A. As a result, whereas the electron density map, >> b-factor >> and geometry for chain A is pretty reasonable for a 3.4 A resolution >> structure, the refinement for the other two chains (B and C) does not behave >> well. Even playing with different weights for geometry, analysing different >> levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map >> for the helical regions is ok, but the electron density map for strands and >> loops of chains B and C are broken along the main chain, B-factors are >> really >> high, and the geometry keeps being distorted. >> >> Right now, the R-factor and R-free are 24.2 and 28.6, respectively. >> >> Any suggestions in how to proceed the refinement? >> And even a more difficult question, how do we report this type of structure? >> How do we deposit those coordinates? We can certainly use chain A as a model >> to perform interesting studies of structure-function relationship, but we >> know >> that chain B and chain C have problems. >> >> Any help will be greatly appreciated. >> >> Regards >> >> Joane >> >> > > > -- > === > All Things Serve the Beam > === > David J. Schuller > modern man in a post-modern world > MacCHESS, Cornell University > schul...@cornell.edu >
Re: [ccp4bb] Quick-soak
Hi Amit, Dr. Dauter has several excellent papers about solving crystal structures with halide ions. Use this search string in PubMed (without the quotes) to retrieve them: "dauter [AUTH] AND halide". The anomalous signal of iodide can be detected using a home x-ray source but the anomalous signal of bromide generally requires a synchrotron source. Good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Thu, 25 Sep 2008, amit sharma wrote: Dear CCP4bbers, I have a protein molecule(~9.0 kDa) that crystallized in the presence of 0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present, I intend to perform heavy metal derivatization. I read some literature which suggested that I could carry out quick soak with 0.5M Sodium iodide. I was wondering if I could soak my crystals with a similar concentration of NaBr and collect some low resolution data at the in-house source? In addition should I try to also introduce other heavy metals? it might be worth mentioning that my protein carries no methionine/cys residues. I have no prior experience in doing this. So, it would be of great help if I could be directed towards literature/protocols pertaining to this. Any advice/suggestions would be greatly appreciated. Thanks in advance -- Amit Sharma
Re: [ccp4bb] difficult MAD dataset
Hi Allison, How sure are you of your spacegroup? Have you tried P212121? It is a much more common spacegroup. How did you look for your systematic absences? It is possible that the systematic absences are also present along the third axis even though all the reflections have not been measured. Scalepack would not show "absences" for the third axis at the end of the logfile if none of the supposed-to-be absent reflections were measured so it could mislead you. good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Thu, 29 Jan 2009, Alison Li wrote: We recently collected a complete 2.5A MAD dataset. However, finding a solution has not been as straightfoward for reasons unclear to us. We would be grateful for any helpful advice or suggestions. The thin plate shaped crystal was grown from a relatively small protein (90 residues). The crystal diffracted well with visible and defined reflections up to ~2.8A range. With both HKL2000 and mosflm, initial indexing indicated orthorhombic unit cell with dimension of 52 x 82 x 100. Systematic absences along a and b axis were observed thus the dataset was scaled in P21212 space group. The unit cell dimension and space group suggested 4 protein chains per ASU. There is a pseudo-translation with 55 % peak at a fractional coordinate of 0.5, 0.5, 0.48. There are 7 methionines per chain. Thus we expect 28 Se per ASU. Mass spectroscopy and fluoresence scan both confirmed successful incorporation of Se-methionine in the crystal. According to xtriage, anomalous signal extended to 3.0A at least in the peak dataset (The table of measurability as a function of resolution is shown below). unused: - 43.0868 [ 0/5 ] bin 1: 43.0868 - 5.3953 [2751/2902] 0.5838 bin 2: 5.3953 - 4.2836 [2893/2905] 0.4575 bin 3: 4.2836 - 3.7425 [2891/2899] 0.2820 bin 4: 3.7425 - 3.4005 [2888/2896] 0.1898 bin 5: 3.4005 - 3.1568 [2864/2898] 0.1222 bin 6: 3.1568 - 2.9708 [2835/2898] 0.0653 bin 7: 2.9708 - 2.8220 [2777/2906] 0.0458 bin 8: 2.8220 - 2.6992 [2714/2902] 0.0226 bin 9: 2.6992 - 2.5953 [2550/2865] 0.0168 bin 10: 2.5953 - 2.5057 [2307/2914] 0.0071 unused: 2.5057 - [ 0/0 ] However, HA search using hkl2map, Autosol, and Autosharp resulted in only 3~4 HA sites. When hkl2map was used, most HA sites had poor CC and Patterson FOM and did not clearly stand out as they normally should. Structural homologs suggest that the protein has a compact single core domain comprised of 4 a-helices. The positions of most HAs are unlikely to be located in the flexible region. If any abnormalies are seen with the dataset, it's during scaling step in HKL2000. Chi2 is unusually high at lower resolution (Chi2 is >3 from 3.5A as shown below) and there is a relatively high percentage of rejections (>1.5 %). Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 5.38 4299.077.349.0 7.886 0.076 0.085 5.38 4.27 2938.752.735.0 5.843 0.083 0.090 4.27 3.73 2314.245.933.5 3.935 0.082 0.084 3.73 3.39 1245.334.428.9 2.838 0.101 0.094 3.39 3.15 658.628.125.9 1.957 0.132 0.127 3.15 2.96 451.927.125.7 1.322 0.157 0.138 2.96 2.82 307.227.126.3 1.001 0.201 0.169 2.82 2.69 253.128.828.1 0.866 0.225 0.193 2.69 2.59 199.331.531.0 0.801 0.262 0.233 2.59 2.50 159.534.734.4 0.688 0.292 0.261 All reflections 1312.939.031.9 2.748 0.100 0.089 Xtriage also complains that there are abnormal intensities at some resolution ranges. Finally, the crystallization requires CdSO4, so we suspect that cadmium ions are incorporated in the crystal. If so, we suspect there may be weak anomalous signal contribution from Cd as well. In summary, we appear to have a complete dataset that shows strong anomalous signal. However it appears that we have overlooked something or there is an unusual crystallographic issue that we are not aware of. Any suggestions will be very much appreciated. Alison Li Graudate student, Dr. Mark Paetzel's group Simon Fraser University, BC, Canada
Re: [ccp4bb] elbow and compound library
Hi Eric, You can try the Dundee prodrg server: http://davapc1.bioch.dundee.ac.uk/prodrg/ Good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 3 Mar 2009, Eric Liu wrote: Hi All, I downloaded a stausporine PDB file and used elbow to generate the cif file for my ccp4 refinement. It gave the following error message: 0:00 Bondise Bondise Bondise Bondise Bondise Bondise Bondise Bondise Bondise B Failed to determine the bonding of a fragment of the molecule. PDB file elbow.stau_pdb.001.pdb written. Bonding file elbow.stau_pdb.001.bonding.py written. Edit bonding file to reflectALTERNATIVELY Re-run eLBOW with the --reel option and the molecule wil be loaded into REEL. Edit the bonds and save the results as "fixed.cif" to allow eLBOW to load the bonding. the desired bond. I guess stausporine was quite complicated molecule. How do I rerun the elbow with reel option? What other programs you can use to generate compound library file? Thanks! Eric
[ccp4bb] Fwd: Crystallography job opportunities at HarkerBio in Buffalo, NY
Hello community. HarkerBIO is structural biology-focused biotechnology company based in Buffalo, NY with a close relationship to the renowned Hauptman-Woodward Medical Research Institute. We are rapidly growing and are seeking to quickly fill a few open crystallography/structural biology positions. Please see the the job description below and submit your resume/CV along with a cover letter to care...@harkerbio.com. thank you. Eric *Scientist/Senior Scientist/Lead/Director: Structural Biology* *Summary* An opportunity to participate in (or, if sufficiently qualified, lead) our structural biology team work. Bring your considerable experience in structure determination and analysis towards advancing client-focused and internal drug discovery and technology development efforts. *Responsibilities & Duties* 1. Lead from the lab: generate structures (protein, protein complexes, etc.) with hands-on involvement throughout the entire process from gene to structure and beyond. 2. Collaborate, coordinate and enable: construct design, expression/purification trials, biochemistry and biophysics – collaboration in tightly matrixed environment is a must. 3. Communicate: work in an integrated team together with project leads, senior staff, and Clients. Write detailed reports, lead teleconference and in-person meetings with Clients. Lead technical meetings and team meetings as appropriate with level. 4. Innovate: proactively work on improvements of existing technologies, discovery of new tools and technologies, contribute to the internal Discovery projects. 5. Grow & Train: advance your knowledge and repertoire of techniques. Learn orthogonal techniques from your Colleagues. Mentor and train others in the art and science of Structural Biology. *Essential Qualifications and Background* 1. Ph.D. in Structural Biology, Biochemistry, or other appropriate field. 2. Expert in preparation and crystallization of proteins, protein assemblies, protein-small molecule complexes, etc. Membrane protein experience is a plus. 3. Mastery of effective data collection at modern synchrotron facilities. 4. Mastery of structure determination techniques and software. 5. Multi-tasking and efficient time/effort management. 6. Excellent interpersonal and communication skills. 7. Ideal candidate has 5+ years of hands-on gene-to-structure experience in a pharma/biotech setting. Other experience levels will be considered – level of responsibility will be scaled accordingly. *Additional Qualifications for Leadership level* 1. Strong leadership experience in matrixed team environment (2+ years leading a team in Pharma or Biotech). 2. Demonstrated capacity to build, motivate, and manage a team of exceptional Structural Biologists. 3. Proven ability to apply orthogonal techniques and knowledge towards solving Discovery challenges (having SPR, NMR, MS, cryoEM, Enzymology experience is highly advantageous). 4. Experience initiating and managing multiple external collaborations. 5. The ability to represent one’s company in discussions with Clients & Partners, good situational and business awareness, high level of presentation and discussion skills – the ability to present complex ideas and results in an elegant, concise, and effective manner is a must. 6. Experience with the budgeting process, practical awareness and application of basic business accounting and cost control skills. -- _____ Eric Larson, PhD HarkerBIO, L.L.C. 700 Ellicott Street, Buffalo, NY 14203 eric.lar...@harkerbio.com -- _____ Eric Larson, PhD HarkerBIO, L.L.C. 700 Ellicott Street, Buffalo, NY 14203 716-898-8658 <(716)%20898-8658> eric.lar...@harkerbio.com
Re: [ccp4bb] cracking crystal
Hi Fengxiale, Have you tried no cryoprotection? 10% PEG4K is probably too low but you may get lucky. Since your crystal is already in PEG, you may try increasing the concentration of the PEG or try just plain ethylene glycol, which may be more gentle on your crystal than sticking it in glycerol. You say you started with 25% glycerol and slowly decreased to 12.5%. Are you doing this by dialysis or by just sequentially transferring your crystal from one solution to the next? Dialysis is much more gentle and you should be going the other direction - i.e. from low conc. of cryoprotectant to high conc (not high to low like you did). How long are you soaking your crystal in the cryoprotectant solution? Do crystals crack immediately or does it take some time? Oftentimes just a quick dip is sufficient. How well does your crystal diffract at room temperature? Perhaps 3.2 A is the limit even in the absence of freezing. Good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 1 Sep 2009, Fengxia Liu wrote: Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
Re: [ccp4bb] Methylation of macromolecular complexes
Hi Peter, How do you know you have crystallized the complex? Perhaps MR is not working because your crystals only contain the small protein. good luck, Eric Hi all, I have a small complex, one component is 13 kDa with structure available and the other is 7 kDa, which could not be able to grew crystals after lots of efforts. It grew crystals after methylation and diffracted to 2.3 A, however, I could not be able to solve the structure using the structure of the big component as a template for molecular replacement, and heavy atoms soaking was not successful. I plan to do selenomethionine expression next. Does the methylation change protein structures a lot? otherwise, why does molecular replacement not work? I would much appreciate any idea and suggestions how to solve the structure using the data and the template avaliable. Many thanks for your help. Peter Date: Tue, 24 Nov 2009 18:27:49 +0100 From: jan...@googlemail.com Subject: [ccp4bb] Methylation of macromolecular complexes To: CCP4BB@JISCMAIL.AC.UK Hi all, I have a question regarding the methylation of macromolecular complexes. Is there any report where a macromolecular complex has been successfully methylated and later on crystallized. Is methylation a method of choice to chemically modify macromolecular complexes where the entropical contributions by lysines might be a cause for not obtaining crystals, or is there any other better ways? thanks, Jan Rash Keep your friends updated— even when you’re not signed in.
Re: [ccp4bb] Slow COOT with Fedora 12
Hi Mark, Is it possible that you used to have coot set up to use the slow computer settings and now you don't. Try adding the slow computer configuration settings to your .coot file. Information can be found at: http://www.ysbl.york.ac.uk/~emsley/coot/doc/chapters/user-manual_8.html#SEC2 02 Good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Thu, 4 Feb 2010, Mark A. White wrote: Hello, Does anyone know why COOT is suddenly as slow as molasses on my Fedora 12 desktop? I had and old version of Coot working after my initial Fedora 12 install (from FC7 after a disk crash). However, now all versions of COOT have the same problem, very slow response and refresh rates. It does not seem to matter whether the directory is local or NFS, even before a PDB file is loaded it slows to a crawl. With a PDB file loaded the interface becomes catatonic. Centering takes several seconds. Any idea what the problem could be? COOT was amazingly fast on the same hardware with FC7, so it must be Fedora12 specific. The glxgears speed seems slow to me. # COOT 0.6.1 coot-Linux-i386-fedora-10-gtk2-python/ coot-Linux-i386-fedora-12-gtk2/ coot-Linux-i386-fedora-4-gtk2/ COOT 0.4.1 coot-Linux-i386-fedora-5 # Hardware TYAN S2865A Tomcat K8E Dual Core AMD Opteron 2.4GHz 2 GB RAM ATI Radeon RV530 (Radeon X1600pro) kernel-PAE-2.6.31.12-174.2.3.fc12.i686 xorg-x11-server-Xorg-1.7.4-1.fc12.i686 # glxgears 8071 frames in 5.0 seconds = 1614.093 FPS 8105 frames in 5.0 seconds = 1620.857 FPS Yours sincerely, Mark A. White, Ph.D. Assistant Professor, Dept. Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Cell. (409) 539-9138 Fax. (409) 747-4745 mailto://wh...@xray.utmb.edu http://xray.utmb.edu http://xray.utmb.edu/~white
Re: [ccp4bb] Displaying electron density in PYMOL
Hi Raja, If you are reading in the map saved from coot, it covers just the AU. Most likely the selection that you are trying to show the mesh around in pymol is outside of the AU. The solution is to use FFT to draw maps around your model (it an option in one of the drop down menus in the FFT CCP4i gui) and use these maps in pymol. And don't forget to add the .ccp4 extension to the map files for reading into pymol. hope that solves your problem. Eric ______ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Thu, 11 Feb 2010, Raja Dey wrote: Hi, I need someone expert in PYMOL. I am getting a trouble to prepare a figure to display electron density for some water molecules. I can see clear density in COOT at sigma level 2.5. But, I could not see the density in PYMOL even at sigma level 1.0 Here is what I have done in PYMOL 1. file…….open ……..pdb and map files. I can see these two objects on the right column. 2. Then I selected the waters for which I want to display the electron density and created an object named ‘wat’ 3. In the command line I wrote ‘isomesh mesh1, 3fo2fc.map, 1.0, wat, carve=1.6’ and it creates an object ‘mesh1’ that shows the electron density of some of the waters (not all) selected in ‘wat’. I tried with other sigma levels, but the problem persists. I could not understand why for some waters electron density did not show up in PYMOL, but it does in COOT. I also noticed the same problem remains for part of this molecule, not just for some waters. I checked the pdb file, which looks fine and have the standard format. Does anyone have any idea, what’s going wrong? Thanks… Raja Your Mail works best with the New Yahoo Optimized IE8. Get it NOW!.
Re: [ccp4bb] Question about modeling SAM into a protein structure
Hi Yuan, LSQ within Coot works quite well for superimposing similar ligands. Are you sure you are selecting the appropriate chain IDs and residue numbers in the LSQ dialog box? It is a rigid body superposition though so it will only get you in the neighborhood (i.e. the adenine and sugar rings should at least superimpose well and then you may have to manually adjust the rest). If you have electron density for the SAH, the 2 ligands are similar enough that you could then use real space refinement to get the SAM to superimpose almost perfectly on SAH. You would have to read a restraint file for SAM (which can be obtained by uploading the SAM coordinates into the PRODRG server) into coot for this though. Good luck, Eric PS - SSM will not work for ligands, as you saw. __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Wed, 3 Mar 2010, Yuan Cheng wrote: > Hi everyone, > I am trying to model a S-adenosylmethionine (SAM) molecule into the active > site of a protein using the SAH (exists in the crystal structure) as the > template. > > What I have already tried but failed so far are > 1)Pymol: I loaded the pdbs of SAM and protein-SAH into pymol and copy SAH > into a separate object. Then I tried to align SAM to SAH, but it didn't work > and pymol said " Executivealign:Mobile selection must derive from one object > only". Does anyone know what this mean? > > 2)Coot: I tried to superpose SAM to SAH in coot. Bot SSM superpose and LSQ > superpose didn't work. when I did SSM superpose, coot said "can't make graph > for SAH structure". when I did LSQ superpose, nothing happened after I run > it. I also tried Extensions> modeling> superpose ligands, nothing happened > too. Does anyone know what's wrong here? > > I know that I can probably manually move SAM to the position of SAH and > change the bond angles to make SAM align to SAH. I am wondering if there is > any program that can help to perform ligand alignment without the need for > manual adjustment. > > Thank you very much for your suggestion! > > Yuan >
Re: [ccp4bb] protein monitoring
Hi Yogi, You can see your peptide on a gel so why can't you "monitor" it by SDS-PAGE? A little time consuming, yes, but then you have the extra benefit of also seeing if there are contaminating proteins in your sample. good luck, Eric ______ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Fri, 28 May 2010, Sollepura Yogesha wrote: 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, I le (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
