Re: [ccp4bb] Off-topic His-Antibody
I have been using an HRP conjugated antibody from Thermo that can detect maybe 3-8ng of HisTagged protein from insect cell lysates. Performance is ok, but with the sensitivity I am not so happy with. How is the sensitivity with the Qiagen antibody? How many picograms you need for a clear band? Does anyone know a good HRP conjugated antibody? Regards, Juha On 26 June 2012 06:59, Luca Jovine luca.jov...@ki.se wrote: We routinely use BSA-free anti-5His mAb from QIAGEN, and it works just fine (at least using media and lysates of both insect and mammalian cells): http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hisantibody.aspx Good luck, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 83 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of D Bonsor [ dbon...@ihv.umaryland.edu] Sent: Monday, June 25, 2012 23:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic His-Antibody Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish. -- Genius may have its limitations, but stupidity is not thus handicapped. -Elbert Hubbard *
Re: [ccp4bb] Off-topic His-Antibody
Our experience essentially agrees with QIAGEN's claim that you can detect proteins in the low nanogram range, i.e. 1-2 ng... so not a major improvement over your current antibody, I'm afraid. They also sell an HRP conjugate version: http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hishrpconjugatekit.aspx but we have never tried it... and now I'll stop here, otherwise you'll start thinking I own QIAGEN shares :-) Luca On Jun 26, 2012, at 09:54 , Juha Vahokoski wrote: I have been using an HRP conjugated antibody from Thermo that can detect maybe 3-8ng of HisTagged protein from insect cell lysates. Performance is ok, but with the sensitivity I am not so happy with. How is the sensitivity with the Qiagen antibody? How many picograms you need for a clear band? Does anyone know a good HRP conjugated antibody? Regards, Juha On 26 June 2012 06:59, Luca Jovine luca.jov...@ki.se wrote: We routinely use BSA-free anti-5His mAb from QIAGEN, and it works just fine (at least using media and lysates of both insect and mammalian cells): http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hisantibody.aspx Good luck, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 83 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of D Bonsor [dbon...@ihv.umaryland.edu] Sent: Monday, June 25, 2012 23:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic His-Antibody Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish. -- Genius may have its limitations, but stupidity is not thus handicapped. -Elbert Hubbard *
[ccp4bb] practical protocol to remove cell culture derived Threonine from the protein
Dear ALL, The threonine is found in the active site of my protein structure from the crystallization in the absence of any threonine containing chemicals. I presume that's why there was no signals detected with the ITC experiment in which I titrate my protein with Threonine. In the in vitro biochemical assay, threonine is one of the substrates in the reconsitituted system. So could anyone offer me a practical protocol to remove the threonine from the protein for further experiment to confirm the binding of Threonine to my protein by ITC. Thank you all. Wenhua Ph. D student in structural biology Paris-Sud XI, France
[ccp4bb] Post doctoral vacancy in structural biology, Oxford (deadline 23.7.)
Dear All, I would like to draw your attention to a post doctoral vacancy in my group, located at the Division of Structural Biology (StruBi), University of Oxford. Your role will be to study the structure of an enveloped virus using electron cryo-microscopy (cryo-EM). Necessary training will be provided. The project involves working only with non-hazardous materials, such as chemically inactivated viruses. The position is funded by the Medical Research Council and is fixed-term until 30 June 2015 in the first instance. Only applications received before 12.00 midday on 23 July 2012 will be considered. Please quote reference 103398 on all correspondence. You will be required to upload a CV and supporting statement as part of your online application. The application form, full job description, and selection criteria are available at http://www.recruit.ox.ac.uk. Please address all informal inquiries about the post directly to me. Juha -- Dr. Juha T. Huiskonen Oxford Particle Imaging Centre Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive, Oxford OX3 7BN http://www.strubi.ox.ac.uk/ Email: j...@strubi.ox.ac.uk Tel.: +44 1865 287 844
Re: [ccp4bb] practical protocol to remove cell culture derived Threonine from the protein
Hi Wenhua Have you tried using 6-8M urea or guanidinium hydrochloride to denature your protein+threonine, then refold by dialysis into a non-threonine-containing buffer? If you are performing ITC, then i guess you already have an efficient activity assay, thus maybe use that as an indicator of successful refoldingcombined with successful crystal formation may give you confidence in a correct, uniform fold too. If your unfolded protein is His-tagged etc and dialysis isn't totally doing the trick, then perhaps immobilising your protein to a His-column might allow you to physically wash away the contaminating threonine. You can then try an on-column refold, or elute and perform dialysis refolding again. Good luck, Rick Salmon On 26 June 2012 10:08, Wenhua Zhang xtal.zh...@gmail.com wrote: Dear ALL, The threonine is found in the active site of my protein structure from the crystallization in the absence of any threonine containing chemicals. I presume that's why there was no signals detected with the ITC experiment in which I titrate my protein with Threonine. In the in vitro biochemical assay, threonine is one of the substrates in the reconsitituted system. So could anyone offer me a practical protocol to remove the threonine from the protein for further experiment to confirm the binding of Threonine to my protein by ITC. Thank you all. Wenhua Ph. D student in structural biology Paris-Sud XI, France
Re: [ccp4bb] practical protocol to remove cell culture derived Threonine from the protein
I don't know the exact reaction your protein is performing, but an alternative would be to try to turnover your threonine substrate by adding the other substrates. The reaction product should more easily leave the active site. You might have to do an additional gelfiltration to get rid of excess cosubstrates. Good luck as well, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard M Salmon Sent: Tuesday, June 26, 2012 1:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] practical protocol to remove cell culture derived Threonine from the protein Hi Wenhua Have you tried using 6-8M urea or guanidinium hydrochloride to denature your protein+threonine, then refold by dialysis into a non-threonine-containing buffer? If you are performing ITC, then i guess you already have an efficient activity assay, thus maybe use that as an indicator of successful refoldingcombined with successful crystal formation may give you confidence in a correct, uniform fold too. If your unfolded protein is His-tagged etc and dialysis isn't totally doing the trick, then perhaps immobilising your protein to a His-column might allow you to physically wash away the contaminating threonine. You can then try an on-column refold, or elute and perform dialysis refolding again. Good luck, Rick Salmon On 26 June 2012 10:08, Wenhua Zhang xtal.zh...@gmail.com wrote: Dear ALL, The threonine is found in the active site of my protein structure from the crystallization in the absence of any threonine containing chemicals. I presume that's why there was no signals detected with the ITC experiment in which I titrate my protein with Threonine. In the in vitro biochemical assay, threonine is one of the substrates in the reconsitituted system. So could anyone offer me a practical protocol to remove the threonine from the protein for further experiment to confirm the binding of Threonine to my protein by ITC. Thank you all. Wenhua Ph. D student in structural biology Paris-Sud XI, France
Re: [ccp4bb] Off-topic His-Antibody
I'll just second, third, and fourth the assessment of the Qiagen anti-pentaHis antibody. I pretty much gave up on anti-His antibodies until we tried this out in the lab and now everyone loves it. Reasonable sensitivity and specificity (we use IR dye-conjugated secondaries and a Li-Cor Odyssey scanner for signal detection) and not bad on cost. And it does detect hexa- and decahistidine tags. Not using HRP anymore since we switched over to IR detection a few years ago. HTH- Brad On Tue, Jun 26, 2012 at 4:17 AM, Luca Jovine luca.jov...@ki.se wrote: Our experience essentially agrees with QIAGEN's claim that you can detect proteins in the low nanogram range, i.e. 1-2 ng... so not a major improvement over your current antibody, I'm afraid. They also sell an HRP conjugate version: http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hishrpconjugatekit.aspx but we have never tried it... and now I'll stop here, otherwise you'll start thinking I own QIAGEN shares :-) Luca On Jun 26, 2012, at 09:54 , Juha Vahokoski wrote: I have been using an HRP conjugated antibody from Thermo that can detect maybe 3-8ng of HisTagged protein from insect cell lysates. Performance is ok, but with the sensitivity I am not so happy with. How is the sensitivity with the Qiagen antibody? How many picograms you need for a clear band? Does anyone know a good HRP conjugated antibody? Regards, Juha On 26 June 2012 06:59, Luca Jovine luca.jov...@ki.se wrote: We routinely use BSA-free anti-5His mAb from QIAGEN, and it works just fine (at least using media and lysates of both insect and mammalian cells): http://www.qiagen.com/products/protein/detection/qiaexpressdetectionsystem/penta-hisantibody.aspx Good luck, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 83 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of D Bonsor [ dbon...@ihv.umaryland.edu] Sent: Monday, June 25, 2012 23:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic His-Antibody Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish. -- Genius may have its limitations, but stupidity is not thus handicapped. -Elbert Hubbard *
[ccp4bb] off topic detection of oligomerisation
Dear ccp4 bulletin board I apologise for off topic question. I wonder if anybody knows of a good method to detect oligomerisation? I suspect an equilibrium intermediate is forming oligomers based on tryptophan fluorescence showing an exposed tryptophan becoming buried in a hydrophobic region. I would like to perform some experiments to prove this. Cross linking is not working for me and neither is SEC HPLC due to technical issues with the urea I think. Are there any other quick and easy techniques that any of you can think of off the cuff? Shifts in equilibrium unfolding curves done at different concentrations will probably show something because of the law of mass action but this is a big job needing many replicates so I wonder if there are any other simple methods available? Thanks Careina
Re: [ccp4bb] off topic detection of oligomerisation
Native PAGE (i.e. BN-PAGE), light scattering (i.e. MALLS) Not quick and easy but could work: AUC (i.e. a sedimentation equilibrium experiment) -Brad On Tue, Jun 26, 2012 at 9:01 AM, Careina Edgooms careinaedgo...@yahoo.comwrote: Dear ccp4 bulletin board I apologise for off topic question. I wonder if anybody knows of a good method to detect oligomerisation? I suspect an equilibrium intermediate is forming oligomers based on tryptophan fluorescence showing an exposed tryptophan becoming buried in a hydrophobic region. I would like to perform some experiments to prove this. Cross linking is not working for me and neither is SEC HPLC due to technical issues with the urea I think. Are there any other quick and easy techniques that any of you can think of off the cuff? Shifts in equilibrium unfolding curves done at different concentrations will probably show something because of the law of mass action but this is a big job needing many replicates so I wonder if there are any other simple methods available? Thanks Careina
Re: [ccp4bb] practical protocol to remove cell culture derived Threonine from the protein
Dear Wenhua, in extension to the post from Herman, you could try to remove the threonine with a separate enzyme, for instance threonine oxidase or amino acid deaminase. Their reaction products probably also bind less tightly. The question is, how fast the release of threonine from your protein is. greetings Gottfried Am Dienstag, dem 26-06-2012 um 11:08 schrieb Wenhua Zhang: Dear ALL, The threonine is found in the active site of my protein structure from the crystallization in the absence of any threonine containing chemicals. I presume that's why there was no signals detected with the ITC experiment in which I titrate my protein with Threonine. In the in vitro biochemical assay, threonine is one of the substrates in the reconsitituted system. So could anyone offer me a practical protocol to remove the threonine from the protein for further experiment to confirm the binding of Threonine to my protein by ITC. Thank you all. Wenhua Ph. D student in structural biology Paris-Sud XI, France
[ccp4bb] Cover pictures
Dear All: Are there any web sites that produce stunning artistic images from pdb files? david -- David M. Mueller Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science Green Bay Road North Chicago, IL 60064 *david.muel...@rosalindfranklin.edu http://lmb-il.org/index.html *Office: 847-578-8606 FAX: 847-578-3240
Re: [ccp4bb] Cover pictures
[Advertisement on] Follow the link and click on her website: http://web.me.com/bosch_lab/jbosch/News/Entries/2012/4/7_Cover_figure_in_Journal_of_Structural_Biology_%28April_2012_issue%29.html In case the link doesn't work then just go to http://web.me.com/bosch_lab and click on the News [Advertisement off] This was all done remotely, so two phone calls and lots of email exchange. It was fun to do it. Jürgen On Jun 26, 2012, at 9:44 AM, David Mueller wrote: Dear All: Are there any web sites that produce stunning artistic images from pdb files? david -- David M. Mueller Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science Green Bay Road North Chicago, IL 60064 david.muel...@rosalindfranklin.eduhttp://david.muel...@rosalindfranklin.edu/ http://lmb-il.org/index.html Office: 847-578-8606 FAX: 847-578-3240 .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] Cover pictures
Never mind my previous email, I misread - I thought Journal Cover figures. What's wrong with PyMol or PovRay DoItYourself ? Jürgen On Jun 26, 2012, at 9:44 AM, David Mueller wrote: Dear All: Are there any web sites that produce stunning artistic images from pdb files? david -- David M. Mueller Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science Green Bay Road North Chicago, IL 60064 david.muel...@rosalindfranklin.eduhttp://david.muel...@rosalindfranklin.edu/ http://lmb-il.org/index.html Office: 847-578-8606 FAX: 847-578-3240 .. 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://web.mac.com/bosch_lab/
[ccp4bb] [OT]: The Ultimate Inferior Beings
This is to inform you that a well-respected member of the crystallographic community has published a totally hilariously absurdly funny Sci-Fi novel. I added a link on my web site, www.ruppweb.org , and no, I said 'well respected', so I did not write it, but maybe you can guess from the pseudonym. Cheers, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - No animals were hurt or killed during the production of this email. -
[ccp4bb] Phaser Fatal runtime error.
Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717
Re: [ccp4bb] pdb sequence search
Hi Ed, Another way to look for point mutations are the sequence clusters available at 100%, 95%, 90%, etc. sequence identity. Here is an example: find mutations for HIV-1 protease: Start with an example protein structure, i.e., 1OHR. On the structure summary page of 1OHR click on the Seq. Similarity tab. Then select the 95% seq. id cluster to retrieve all proteins with = 95% seq. identity to the query structure: http://www.rcsb.org/pdb/explore/sequenceCluster.do?structureId=1OHR Peter Rose RCSB PDB -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Friday, June 22, 2012 6:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pdb sequence search Silly question. Say I want to find every structure in the PDB with the exact sequence or with perhaps 1-2 mutations. I know of two ways of doing this. 1. Go to NCBI BLAST and run the sequence against the PDB subset. The resulting list will have identities listed, so manual parsing is doable if there aren't too many hits. 2. PDB and PDBe both have the search by sequence features. Trouble is the default E value seems to be tailored to poor sequence identity (which makes sense if you looking for potential MR models). Sure, I can reduce the target E value, but it's a little cumbersome and I have no idea what the target level should be so that I don't get any 50% identical sequences yet not miss single/double mutants. Wouldn't it be nice if one could use the sequence identity cutoff/query coverage instead? Much more comprehensible than the E-value. Is there a search engine that does that? Seems like a fairly common need, and perhaps I just can't find on PDB website. Thanks in advance for any suggestions, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
[ccp4bb] promega protev
I apologise for off topic question. I wonder if anybody knows a good buffer/condition for promega protev? I need to remove his-tag from my sensitive mamalian protein which does not like the NaCl concentration less than 300mM. Best, Jahan
[ccp4bb] relative intensity of ice rings
Hi, all, I thought I could easily find a reference to comment upon the relative intensity of rings in an image due to diffraction by polycrystal ice, but no luck googling for that. A reference that would contain a picture (with visual relative intensities) would be even better. Of course absolute intensity depends on the amount of ice, but a relative scale is what I am looking for now. Yours, Jorge
Re: [ccp4bb] Phaser Fatal runtime error.
Don't know where the exact problem is. However, it is definitely possible to use a Crysalis-Scala-Truncate-Phaser pipeline without runtime errors. I have done a few times. I am sure you will be able to get help from Agilent people. If not, feel free to get back to me. Best Roberto On 26 Jun 2012, at 18:34, Stephen Carr wrote: Dear CCP4bb I have collected a data-set using the supernova x-ray generator from Agilent and taken the mtz file generated by the data processing software in crysalis pro forward for structure solution. The data collection was straight forward and the software seemingly processed the data successfully - space-group P2221, overall Rmerge 9%, I/sigmaI 11, redundancy 4.5 etc. Truncate converted the intensities to structure factors with no problems, but when I tried to use the data for molecular replacement with Phaser it produced the following error: FATAL RUNTIME ERROR: Reflections are not a unique set by symmetry I'm not sure how to proceed from here as other programs in the suite do not seem to detect this problem. Also when this error has been mentioned in the past on the bb it was with a data set collected on a Bruker home source and the data processed with Denzo/scalepack, and the suggested solution was to use the Bruker software to process the data. I am currently attempting to reprocess the data with mosflm, but that is likely to be the subject of another post! Any suggestions will be gratefully received. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.uk
Re: [ccp4bb] relative intensity of ice rings
Maybe figure 4 in Viatcheslav Berejnov et al. Vitrification of aqueous solutions J. Appl. Cryst. (2006). 39, 244–251 ? JORGE IULEK wrote: Hi, all, I thought I could easily find a reference to comment upon the relative intensity of rings in an image due to diffraction by polycrystal ice, but no luck googling for that. A reference that would contain a picture (with visual relative intensities) would be even better. Of course absolute intensity depends on the amount of ice, but a relative scale is what I am looking for now. Yours, Jorge
Re: [ccp4bb] relative intensity of ice rings
I think an appropriate reference is Bragg (1921) http://dx.doi.org/10.1088/1478-7814/34/1/322 who compared various possible crystal structures to the relative strength of the reflections from ice powder measured by Dennison (1921) http://dx.doi.org/10.1103/PhysRev.17.20. However, as Bragg noted Dennison's intensities don't agree all that well with those you would expect from what we now know is the structure of hexagonal ice (Ih). It is possible that Dennison's preparation (plunge-cooling pure water in a capillary) actually created some cubic ice (ice Ic) along with his hexagonal ice (ice Ih). The high intensity he saw for the middle line of the triplet we normally see for true hexagonal ice is consistent with this. Cubic ice is actually more commonly seen in MX diffraction patterns than hexagonal ice (in my experience). However, you do have to be very careful about what you mean by intensity. Are you talking about photons/pixel? Photons/spot? Photons integrated over a powder_ring? All these will be different relative numbers. I'm not sure if they knew about Lorentz factors yet in 1921 There is no mention of correcting for them in either paper. Anyway, if you are after the true hexagonal ice ring intensities, I would advise taking the following PDB file: CRYST1 4.511 4.511 7.346 90.00 90.00 120.00 P 63/m m c SCALE1 0.221680 0.127987 -0.00 -0.0 SCALE2 -0.00 0.255974 -0.00 0.0 SCALE3 0.00 -0.00 0.136129 -0.0 ATOM 1 O WAT A 1 0.000 2.604 0.457 1.00 0.00 O ANISOU 1 O WAT A 1 603 630 172 302 0 0 O ATOM 2 H WAT A 1 0.000 2.604 1.308 0.50 0.00 H ANISOU 2 H WAT A 1 510 510 56 255 0 0 H ATOM 3 H WAT A 1 0.000 3.432 0.148 0.50 0.00 H ANISOU 3 H WAT A 1 487 361 163 185 199 95 H Calculate structure factors from it, add up F^2 of same-resolution indicies and plot them out that way. remember, the square of a structure factor is proportional to the integrated intensity of a single-crystal spot, which is not the same thing as a powder ring intensity. The relationship was described most recently by Norby (1997) http://dx.doi.org/10.1107/S0021889896009995 Which I paraphrase as: for a flat detector, the average intensity of a pixel in a powder ring is given by: Ipix = k*p*sum(F^2)*omega/sin(theta)/sin(2*theta) where Ipix is the recorded value of one pixel, 'k is a resolution-independent scale factor, p is the polarization factor (see Holton Frankel 2010), F is the structure factor of an hkl index that falls on the ring (there can be more than one, hence the sum), omega is the solid angle subtended by the pixel and theta is the Bragg angle. For the above PDB, I get: d sum(F^2) 3.907 121 3.673 156 3.449 111 2.676 88.5 2.255 111 2.075 153 1.953 62.9 1.922 84.2 1.888 62.5 1.836 4.33 1.725 47.8 1.662 1.84 1.527 96.7 1.477 42.8 1.448 39.5 1.375 78.9 1.370 34.6 HTH, -James Holton MAD Scientist On Tue, Jun 26, 2012 at 2:34 PM, Edward A. Berry ber...@upstate.edu wrote: Maybe figure 4 in Viatcheslav Berejnov et al. Vitrification of aqueous solutions J. Appl. Cryst. (2006). 39, 244–251 ? JORGE IULEK wrote: Hi, all, I thought I could easily find a reference to comment upon the relative intensity of rings in an image due to diffraction by polycrystal ice, but no luck googling for that. A reference that would contain a picture (with visual relative intensities) would be even better. Of course absolute intensity depends on the amount of ice, but a relative scale is what I am looking for now. Yours, Jorge
Re: [ccp4bb] The effect of His-tag location on crystallization
Google yields amongst others: His-tag impact on structure Acta Cryst. (2007). D63, 295301 ...quäl dich. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of weliu Sent: Tuesday, June 26, 2012 6:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] The effect of His-tag location on crystallization Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
[ccp4bb] Post doctoral vacancy in structural biology, Singapore
Protein Biophysics Laboratory Several positions at postdoctoral, research associate and research assistant levels are available now in Protein Biophysics Laboratory, National University of Singapore. The successful postdoctoral candidate should demonstrate a strong motivation in scientific research; and hold a PhD in protein biophysics or/and structural biology. Both fresh PhD and experienced PhD researcher will be considered. Salary and benefits will be commensurable to qualifications and working experience. Interested individuals can send their applications, academic transcripts, curriculum vitae to A/P Song Jianxing at bc...@nus.edu.sg; Department of Biological Sciences, National University of Singapore. Only shortlisted candidates will be contacted for interview.
