Re: [ccp4bb] His tag on membrane protein
Hi Deliang, My own experience with two membrane proteins suggests that His-tag issues pertaining to soluble proteins also seem to apply to membrane proteins. In one case, for example, cleaving the N-terminal His-tag improved the solubility of the protein dramatically. Interestingly, expression levels of the protein without the His-tag were much lower than with it! In the other case, we have a construct with a His-tag at the N-terminus and another at the C-term. Both behave well when it comes to solubility and stability but we thus far only have diffracting crystals from the construct with the C-term His-tag. However, we have not yet tried to remove the tags from those two constructs to see whether that improves crystallizability. My feeling is that if you do not have solubility/stability/aggregation problems with your His-tagged membrane protein, then there is no reason to expect lower success rates in crystallization trials. Screen with it and hope for the best! By the way, there is a recent paper on the effects of His-tags on crystal structures of soluble proteins. It's worth a look. Carson M et al. His-tag impact on structure. Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):295-301. Best wishes Savvas Savvas N. Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html Quoting deliang [EMAIL PROTECTED]: Hi there, I purified a membrane protein with traditional His-tag on the C terminal. Before crystallization, I wonder how this tag may affect the result. Does anyone have experience that the removel of this tag may improve the result or not? or can provide some references which may give some statistic, like how many membrane proteins have been crystallized with or without His-tag? Thanks so much. Deliang
Re: [ccp4bb] Anisotropic data - How to deal with ?
Concerning what Pierre asked, one point to remember at structure refinement is to test and probably use TLS tensors. I had one case with serious anisotropy in the data due a libration axis parallel to a crystallographic axis. TLS lowered dramatically the R's and maps were neatly better. Concerning one point raised by Anastassis: Think of a 1-2 micron thin plate, very well ordered, and an excellent almost matching micro-beam: shooting 'edge-on' you get a diffracting volume 'along' the plate and data to - say - 2.5 A. shooting 'face-on' you get a much lesser diffracting volume 'through' the plate and data to - say - 3.2 A. (if you shoot too much to get 2.5 you kill the crystal in no-time) I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection and also some other concerns about beam homogeneity in its cross section. How serious is this nowadays ? Can processing programs easily overcome, in a certain mounting, the fact that not all crystal orientations have the same number of unit cells exposed to x-rays ? What about inhomogeneities at the beam ? I understand that technical difficulties may lead you to exposed your crystal partially to the beam, etc..., but how hard should we care about this (how much effort to avoid this) ? Jorge
Re: [ccp4bb] To bathe or not to bathe.
The main reason was related to absorption. If you didn't completely bathe the crystal in the xray beam, then the diffracting volume of the crystal would be different during the data collection, and thus, scaling would be inaccurate, especially when there was radiation damage. This was especially true when you weren't sure that the crystal was well-centered in the xray beam (in a cryostat, and therefore not visible). We typically collected highly redundant data to help compensate for this. We also used to correct for absorption by assigning Bragg indices to the crystal and making precise measurements of crystal dimensions. Scaling programs are now more extensive, and include options to calculate a pseudo-absorption surface. In principle, if you have a beam that is ALWAYS smaller than the crystal, then the same crystal volume is illuminated by the xray beam, and will minimize scaling errors. Bernie Santarsiero On Fri, November 23, 2007 4:34 pm, Jim Pflugrath wrote: It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
Re: [ccp4bb] To bathe or not to bathe.
It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
[ccp4bb] His tag on membrane protein
Hi there, I purified a membrane protein with traditional His-tag on the C terminal. Before crystallization, I wonder how this tag may affect the result. Does anyone have experience that the removel of this tag may improve the result or not? or can provide some references which may give some statistic, like how many membrane proteins have been crystallized with or without His-tag? Thanks so much. Deliang
Re: [ccp4bb] Summary: Symmetry of crystallographic stereographic projections?
Doesn't POLARRFN have this option too (i.e. to print all symmetry related peaks) ? Maybe not through the gui but most likely in command line mode. Boaz - Original Message - From: Eleanor Dodson [EMAIL PROTECTED] Date: Friday, November 23, 2007 12:37 Subject: Re: [ccp4bb] Summary: Symmetry of crystallographic stereographic projections? To: CCP4BB@JISCMAIL.AC.UK A bit late but I didnt answer in time. This is one of the cases where I still wheel out ALMN to do the self rotation. It generates ALL symmetry peaks and allows you to select which axis you want to take as the polar axis. (ncode = 1 c*, ncode = 2 a*, ncode = 3 b* and so on) And the program output also makes an attempt to explain what it is doing! A script #!/bin/csh -f # almn \ hklin /y/people/ccp4/projects/mao/maon5_all_scaleit1.mtz \ MAPOUT /y/work/ccp4//almn.map \ eof SELF 3 25 RESO 10 3.5 TITL Jean CRYS file 1 orth 1 flim 1 10 LABI FILE 1 F=F_natlo LIMIT 0 180 5 1 ! Beta limit 90 because of symmetry ! If in doubt set Beta limit 180.. FIND 5 40 NOPR MAP END eof Dirk Kostrewa wrote: Dear CCP4ers, I've asked you about symmetry in stereographic projections of self-rotation functions, because I have in a monoclinic space group with beta=97 a peak for a NCS 7-fold axis at Phi=83, Psi=90, Kappa=51.4. In this self-rotation function, calculated with GLRF, the monoclinic b-axis is oriented from south-pole to north-pole. Thus, with Psi=90, the 7-fold lies in the a,c-plane, and I was wondering wether it points along the c-axis by some symmetry element, since 83=180-97. Pierre Rizkallah pointed to the fact that the self- rotation function is calculated in Patterson space which has an inversion centre, and Ian Tickle has pointed me to the POLARRFN documentation that discusses some of these symmetries. From this, I can, in my case, construct the following symmetry-equivalent positions: (1) Original Peak: Phi, Psi, Kappa --- 83, 90, 51.4 (2) General equivalent position: 180+Phi, 180-Psi, -Kappa --- 263, 90, -51.4 (3) Crystallographic Dyad || Poles: 180+Phi, Psi, Kappa --- 263, 90, 51.4 (4) (3) combined with (2): Phi, 180-Psi, -Kappa --- 83, 90, -51.4 Thus, none of these combinations gives a Phi-angle of 97 degrees. However, meanwhile Liang Tong, the author of GLRF, explained to me, that in his convention, a positive Phi angle goes from +X (=a) to -Z (=-c*), which means that Phi=83 points really along the (-)c- axis (thus confirming my initial hypothesis). 7-fold / / / b-- a /| / | / | / | c c* So, many thanks to all of you who replied and helped me to solve this puzzle! I would still be interested in a textbook or paper discussing symmetry in stereographic projections, though ... Best regards, Dirk. Am 22.11.2007 um 14:40 schrieb Dirk Kostrewa: Dear CCP4ers, does any of you have a good reference describing the symmetry of crystallographic stereographic projections? There is a lot of literature describing rotational symmetry in Eulerian angular space, but I'm not aware of any for polar angles. In particular, I've calculated a self-rotation function for a crystal in space group C2 with a monoclinic beta-angle of 97 degrees in a convention where Phi is the angle from the x-axis (=a-axis) and Psi is the angle from the monoclinic b-axis. I get a beautiful peak for a seven-fold rotation axis at Phi=83 degrees and Psi=90 degrees. I think that the seven-fold NCS-axis should point along the crystallographic c- axis, but then I would expect Phi=97 and Psi=90. Presumably, there must be a symmetry with the term Phi' = 180-Phi, but I would like to _know_ it. I would be grateful for any pointer to a good reference describing symmetry in crystallographic stereographic projections, also for future cases. Best regards, Dirk. *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: [EMAIL PROTECTED] *** *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: [EMAIL PROTECTED] *** Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the
Re: [ccp4bb] [ccp4]: Problems with ccp4bb
Dear Lucas and All the jiscmail team are having problems with mail sent to yahoo accounts. I received this from the jiscmail helpline on 2 Nov. We are having issues delivering to Yahoo addresses and are actively trying to resolve this as quickly as possible. On our tests, the JISCmail emails are being diverted to the spam folder and being delivered in a tardy fashion. This appears to be due to changes that Yahoo have made to their service. They have been in contact with yahoo, and filled in a large report on the matter as yahoo's request, but still there has been no movement on the issue. In the short term, if you are experiencing problems, it may be best to subscribe from a different account. Charles Ballard CCP4 ps - this is bcc'ed to all the subscribed address, so apologies to those who receive multiple copies. On 22 Nov 2007, at 19:18, Lucas Bleicher wrote: Hi! I am subscribed to the ccp4bb mailing list in this mail account. Strangely, I have not received any e-mail from the list in the last week. They're not going to my spam folder either. I have tried to re-subscribe using the web page, but got no response. What can be happening? Thanks in advance, Lucas Bleicher Abra sua conta no Yahoo! Mail, o único sem limite de espaço para armazenamento! http://br.mail.yahoo.com/
[ccp4bb] Summary: Symmetry of crystallographic stereographic projections?
Dear CCP4ers, I've asked you about symmetry in stereographic projections of self- rotation functions, because I have in a monoclinic space group with beta=97 a peak for a NCS 7-fold axis at Phi=83, Psi=90, Kappa=51.4. In this self-rotation function, calculated with GLRF, the monoclinic b-axis is oriented from south-pole to north-pole. Thus, with Psi=90, the 7-fold lies in the a,c-plane, and I was wondering wether it points along the c-axis by some symmetry element, since 83=180-97. Pierre Rizkallah pointed to the fact that the self-rotation function is calculated in Patterson space which has an inversion centre, and Ian Tickle has pointed me to the POLARRFN documentation that discusses some of these symmetries. From this, I can, in my case, construct the following symmetry-equivalent positions: (1) Original Peak:Phi, Psi, Kappa --- 83, 90, 51.4 (2) General equivalent position: 180+Phi, 180-Psi, -Kappa --- 263, 90, -51.4 (3) Crystallographic Dyad || Poles: 180+Phi, Psi, Kappa --- 263, 90, 51.4 (4) (3) combined with (2):Phi, 180-Psi, -Kappa --- 83, 90, -51.4 Thus, none of these combinations gives a Phi-angle of 97 degrees. However, meanwhile Liang Tong, the author of GLRF, explained to me, that in his convention, a positive Phi angle goes from +X (=a) to -Z (=-c*), which means that Phi=83 points really along the (-)c-axis (thus confirming my initial hypothesis). 7-fold / / / b-- a /| / | / | / | cc* So, many thanks to all of you who replied and helped me to solve this puzzle! I would still be interested in a textbook or paper discussing symmetry in stereographic projections, though ... Best regards, Dirk. Am 22.11.2007 um 14:40 schrieb Dirk Kostrewa: Dear CCP4ers, does any of you have a good reference describing the symmetry of crystallographic stereographic projections? There is a lot of literature describing rotational symmetry in Eulerian angular space, but I'm not aware of any for polar angles. In particular, I've calculated a self-rotation function for a crystal in space group C2 with a monoclinic beta-angle of 97 degrees in a convention where Phi is the angle from the x-axis (=a-axis) and Psi is the angle from the monoclinic b-axis. I get a beautiful peak for a seven-fold rotation axis at Phi=83 degrees and Psi=90 degrees. I think that the seven-fold NCS-axis should point along the crystallographic c-axis, but then I would expect Phi=97 and Psi=90. Presumably, there must be a symmetry with the term Phi' = 180-Phi, but I would like to _know_ it. I would be grateful for any pointer to a good reference describing symmetry in crystallographic stereographic projections, also for future cases. Best regards, Dirk. *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: [EMAIL PROTECTED] *** *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: [EMAIL PROTECTED] ***
Re: [ccp4bb] His tag on membrane protein
Hi Deliang, We have crystallized the complex cytochrome b6f with an extension of His6 (no linker) at the c-terminal of cytochrome f (Stroebel, 2003). The His extension takes part to the crystal contacts. The electron density is visible but not very well defined. We have not tried to remove the His6. Many other structure of membrane protein have used His-tag, but still not enough to make sound statistics. Daniel deliang a écrit : Hi there, I purified a membrane protein with traditional His-tag on the C terminal. Before crystallization, I wonder how this tag may affect the result. Does anyone have experience that the removel of this tag may improve the result or not? or can provide some references which may give some statistic, like how many membrane proteins have been crystallized with or without His-tag? Thanks so much. Deliang
