Re: [ccp4bb] sfall bug?
Eleanor, Thanks for the suggestion. I changed atom numbers to 1 and 2 and residue numbers to 1 and 2. The behavior is identical. Thanks! Jens On Tue, 2015-07-07 at 06:48 +0100, Eleanor Dodson wrote: I wonder if this is due to the late residue number. Could you try again reducing that to something smaller. I seem to remember SFALL stores a flag to recall which residue contributed to which density and there could be a limit on its size. Will test when I get near a working system Eleanor On 6 July 2015 at 22:53, Jens Kaiser kai...@caltech.edu wrote: All, We seem to have stumbled upon a problem in sfall. The two attached pdb files are nearly identical, except the coordinates and b-factors for the two atoms are swapped. When calculating Fs with sfall, we get drastically different mtz files. Upon calculating the corresponding Fcalc maps, it seems that the second atom in a.pdb gets ignored, whereas both atoms in b.pdb are included. There is nothing obvious in the log files to hint to what is happening (i.e. both files state Number of atoms input= 2 Number of atoms in sort = 2 Number in density generation = 2 Number completely within fft box = 2 Minimum B = 5.91 Maximum B = 5.97 Average B = 5.94 We observed this behavior on mac and on Linux. Cheers, Jens
Re: [ccp4bb] sfall bug?
Maybe something to do with the SCALE tag? Minglei On Jul 6, 2015, at 11:02 PM, Jens Kaiser kai...@caltech.edu wrote: Eleanor, Thanks for the suggestion. I changed atom numbers to 1 and 2 and residue numbers to 1 and 2. The behavior is identical. Thanks! Jens On Tue, 2015-07-07 at 06:48 +0100, Eleanor Dodson wrote: I wonder if this is due to the late residue number. Could you try again reducing that to something smaller. I seem to remember SFALL stores a flag to recall which residue contributed to which density and there could be a limit on its size. Will test when I get near a working system Eleanor On 6 July 2015 at 22:53, Jens Kaiser kai...@caltech.edu wrote: All, We seem to have stumbled upon a problem in sfall. The two attached pdb files are nearly identical, except the coordinates and b-factors for the two atoms are swapped. When calculating Fs with sfall, we get drastically different mtz files. Upon calculating the corresponding Fcalc maps, it seems that the second atom in a.pdb gets ignored, whereas both atoms in b.pdb are included. There is nothing obvious in the log files to hint to what is happening (i.e. both files state Number of atoms input= 2 Number of atoms in sort = 2 Number in density generation = 2 Number completely within fft box = 2 Minimum B = 5.91 Maximum B = 5.97 Average B = 5.94 We observed this behavior on mac and on Linux. Cheers, Jens
Re: [ccp4bb] DNA RNA annealing problem
Le Mardi 7 Juillet 2015 14:03 CEST, Almudena Ponce Salvatierra maps.fa...@gmail.com a écrit: Hello Do you have full complementarity of DNA and RNA ? Does the difference in lenght (17 nt/19 nt) mean that you have a bulge on the DNA when the duplex is formed ? In short, what is the Tm of this duplex at these strand and salt concentrations? If the Tm is 35 °C, then room temperature may be a bit hot depending on where you are in this month of July. Also, what was the temperature of the gel during migration ? Do you have access to ITC ? If yes, we have found that this is a perfect technique (1) to check that you have formation of the duplex (basepair formation generates a lot of heat), (2) that you are using 100 % of each strand (or may be less...), (3) that you can stop injecting the second strand when the stoichiomertic ratio is 1/1 (no strand in excess: perfect for crystallization) and, finally, (4) that you can retrieve the sample from the cell, concentrate it and make crystallization drops. See: Da Veiga C., Mezher J., Dumas P., and Eric Ennifar (2015).Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes. In Nucleic Acid Crystallography : Methods and Protocols. (Ennifar E., ed..) in press. Humana Press. NY. Abstract I hope this can be useful. Philippe Dumas 2015-07-03 17:14 GMT+02:00 ChenWeiFei weife...@outlook.com: Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] OT: mapping PDB to mmCIF data quantities
On Tuesday, 07 July, 2015 13:05:55 Phil Jeffrey wrote: I'm updating some code to have limited mmCIF/PDB format interoperability and have hit a snag. While I can infer the connection between some data items in the PDB header REMARK and the items in mmCIF I can't definitively deduce some others. In particular the mapping of REMARK 2 RESOLUTION seems a little ambiguous and the dictionary documentation doesn't help in this regard. So far as I know, anything that begins with REMARK is not guaranteed to follow any standardized convention. Different programs fill in different things here, and depositors can add new stuff. The current PDB documentation states: REMARK 2 states the highest resolution, in Angstroms, that was used in building the model. As with all the remarks, the first REMARK 2 record is empty and is used as a spacer. Used in building the model is nicely ambiguous, so I doubt that you can map it uniquely to any single value reported by some particular program. Ethan Does anyone know where to find an explicit mapping of one data field to another between the two formats ? (I don't expect there to be a data field in the PDB header for everything in mmCIF but I do for the reverse case). Thanks Phil Jeffrey Princeton -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg MS 357742, University of Washington, Seattle 98195-7742
Re: [ccp4bb] determine NCS operators from PDB coordinates
Well done. Sorry for the many typos, but they don't seem to have slowed you down any! eab On 07/07/2015 11:25 AM, Tobias Beck wrote: Dear all, Thanks for your helpful emails. Here is a short summary: I followed Edward Berry's suggestions. Unfortunately I could not use any superposition to the new structure, as suggested by some, since the structures are too different. I used lsqman and mama to obtain the sperical polars for my twofold as pointed out by Ed. After this, I rotated my structure in two steps based on these angles, but using rotation matrixes in the form cos_theta -sin_theta0 sin_thetacos_theta0 0 0 1 as pointed out by Ed, to align the twofold axis in my structure to a twofold axis in the new space group. That was done in pdbset (I did not need to consider translation in my case). Thanks again for helping me! Best wishes, Tobias. On Fri, Jun 19, 2015 at 4:54 PM, Edward A. Berry ber...@upstate.edu mailto:ber...@upstate.edu wrote: A number of superposition programs allow to superimpose specified atoms (such as CA). Once you get the operator, comparing two different operators is not a job for a conventional superposition program, since you are superimposing a line on a line which has the extra degree of freedom- rotation about the line. If you express the operator in spherical polar coordinates, which the superposition program may provide or you can get from the matrix using ccp4 rotmat, you should be able to work out the relation between the axes. Using the Uppsala sofware factory programs: #This is superimposing parts of chains C, A, B on P, N, O (and vice versa - it's proper 2-fold) lsqman -b eof chain_mode original re m1 cbc596.pdb exp m1 C20-370 P20-370 A20-200 A250-400 B30-200 B250-400 N20-200 N250-400 O30-200 O250-400 m1 P20 C20 N20 N250 O30 O250 A20 A250 B30 B250 save m1 m1 ncsasc01.odb quit eof This prints the operator and saves it in ncsasc01.odb* - The 2010 atoms have an RMS distance of0.130 A - RMS delta B = 15.367 A2 - Corr. coeff. = 0.6601 - Rotation: -0.834671 tel:0.834671 -0.550747 -0.001321 tel:0.550747%20-0.001321 --0.550747 tel:0.550747 0.834660 0.004400 tel:0.834660%20%200.004400 --0.001321 0.004400 -0.89 tel:0.89 - Translation :129.38438.428 171.594 --- Now use mama in an off-label way to convert to polar coordinates: mama overlap ncs ncsasc01.odb gives: --- - RT-OP 1 =-0.8346710 tel:0.8346710 -0.5507473 tel:0.5507473 -0.0013208129.384 - -0.5507473 tel:0.5507473 0.8346604 tel:0.8346604 0.0044000 38.428 - -0.00132080.0044000 -0.894 tel:0.894 171.594 - Determinant of rotation matrix 1.00 - Column-vector products (12,13,23) 0.000.000.00 - Crowther Alpha Beta Gamma 106.709 179.736 73.291 - ***Spherical polars Omega Phi Chi 90.132 -73.291 180.000*** - Direction cosines of rotation axis 0.287514 tel:0.287514 -0.957774 -0.002303 tel:0.957774%20%20%20-0.002303 - X-PLOR polars Phi Psi Kappa *undefined* *undefined* 180.000 - Lattmann Theta+ Theta2 Theta- -180.000 179.736-146.582 - Rotation angle 180.000 - * if you use the .odb file outside of the USF software, be aware the matrix is the transpose (or more accurately it is written by columns). In ccp4 this is taken care of withthe keyword odb. On 06/19/2015 09:07 AM, Tobias Beck wrote: Dear all, I have a PDB file that contains NCS in the asymmetric unit, probably point group D3. 1.) What program is recommended for determining the symmetry operators from PDB coordinates? I found findncs, but this uses only heavy atom coordinates (I could probably use just the sulfurs from the PDB as a work around). 2.) Then I would like to compare the PDB file to a related structure. Here I would like to align the symmetry operators determined above with symmetry elements found in a different space group, for example align the twofold axis from NCS with a twofold axis in found in a particular space group. What is a good way to go about this? I am aware that NCS is used in programs as restraints during refinement, but here I am interested in obtaining the NCS symmetry operators and aligning them to symmetry elements present in a new space group. Maybe I am overlooking an obvious solution. Any help is greatly appreciated. Thanks and best wishes, Tobias. -- ___
Re: [ccp4bb] sfall bug?
Hi Jens, I do get the same results when running sfall xyzin a.pdb hklout a.mtz mapout a.map EOF MODE SFCAL XYZIN ATMMAP RESO 100 2 SFSG P1 SYMM P21 NOSC END ie. enforcing P1 for the structure-factor calculation. The density map (on MAPOUT) is on top of the atoms as expected. Unfortunately, the output MTZ file then has a hemisphere of data (P1) ... which is probably not what you want. If I remove the SFSG P1 card (ie let SFALL decide on the SG for SF-calculation itself): the ATMMAP generated for a.pdb is just wrong - it is missing density for the 35.753 7.581 -12.182 1.00 5.91 atom. As Kay says: maybe an issue with atom sorting ... or in the PDB reading via MMDB? It would be interesting to see if this issue is only for SFSG P21 or also for other non-P1 cases, eg. P212121. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] sfall bug?
Just a few observations: I ran echo MODE SFCALC XYZIN\\n SYMM P21\\n RESO 30 3 | sfall xyzin a.pdb hklout temp.mtz a.log and echo MODE SFCALC XYZIN\\n SYMM P21\\n RESO 30 3 | sfall xyzin b.pdb hklout temp.mtz b.log and looked at a.log and b.log They differ only as follows: a.log has First 10 atoms of atsort - orthog coordinates 16204FE1 ICS 6496 10.0180 -7.5680 56.1090 1.005.976 fractional coordinates 0.38411-0.05791 0.55929 First 10 atoms of atsort - orthog coordinates 16289FE1 ICS 7496 35.7530 7.5810-12.1820 1.005.916 fractional coordinates 0.38376 0.05800-0.12143 First 10 atoms of sorted file in asym unit - 20.61588 0.44071 0.44209105.97 1.00 6 ***ZZZ END 10.38376 0.87857 0.05800205.91 1.00 6 ***ZZZ END First atom of sorted file in atsort 20.61588 0.44071 0.44209105.97131.00 ***ZZZ END whereas b.log has First 10 atoms of atsort - orthog coordinates 16204FE1 ICS 6496 35.7530 7.5810-12.1820 1.005.916 fractional coordinates 0.38376 0.05800-0.12143 First 10 atoms of atsort - orthog coordinates 16289FE1 ICS 7496 10.0180 -7.5680 56.1090 1.005.976 fractional coordinates 0.38411-0.05791 0.55929 First 10 atoms of sorted file in asym unit - 10.38376 0.87857 0.05800105.91 1.00 6 ***ZZZ END 20.61588 0.44071 0.44209205.97 1.00 6 ***ZZZ END First atom of sorted file in atsort 10.38376 0.87857 0.05800105.91131.00 ***ZZZ END What I don't understand is why the sorted lists are different? Sorting should make the resulting lists look the same, to my limited understanding. What is peculiar about the coordinates of these atoms is that there x coordinate in fractional units is almost the same, and their y coordinate is mirrored at the origin.Maybe that could play a role. Kay
Re: [ccp4bb] paired refinement
Chiming in late with a follow-up question: On the other hand in paired refinement, if adding the data improves the structure as measured by Rfree in a zone excluding the added data, then it is hard to deny that that data are worth including. Is it correct to think that model geometry would also be valuable at this point? If adding reflections lead to a model with more reasonable average bond angles, reduced clashes, etc., that would indicate that the added reflections have improved the refinement, no? The geometry stats should also be completely independent from the crystallographic stats. Shane Caldwell McGill University On Fri, Jul 3, 2015 at 2:56 AM, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: On Thu, 2 Jul 2015 13:25:07 -0400, Edward A. Berry ber...@upstate.edu wrote: My take on this- No one has been willing to specify a cutoff (and probably there is no rigorous way to mathematically define the cutoff) and say If CC* (or CCfree or whatever) is below X then it will not improve your structure, if above X then it will. the electron microscopy community uses a similar measure (FSC, Fourier Shell Correlation) as CC1/2. They follow their Gold Standard if they cut their data at FSC=0.143 . Mathematically, CC1/2=0.143 is equivalent to CC*=0.5 . So researchers of that community _do_ specify a cutoff, and by calling it Gold Standard they cast it in stone. Very helpful because probably no reviewer ever calls a Gold Standard into question. Probably depends among other things on how strong the lower resolution data is, how good the structure is without the added data. the latter point is crucial: a structure that is good can make use of higher-resolution data than a structure that is not properly refined. That is different from the situation in electron microscopy, where the phases are obtained experimentally. This is why an X-ray structure at an early stage of iterative refinement/fitting may possibly not be improved by the weak high-resolution data , and why the paired refinement should be done during the end game of refinement. But as long as CC1/2 is statistically significant it may improve a very good model even if CC*0.5 (see Bublitz et al IUCrJ 2, 409-420 (2015) for an example). To find out how close the structure to the data is, it helps to compare CCwork and CC*. An arbitrary cutoff (like CC*=0.5) is thus not sensible in all situations; it may serve as a rule of thumb though since the difference in resolution limit is not large anyway: CC*=0.5 and CC*=0.3 usually differ by (say) 0.1A only. On the other hand in paired refinement, if adding the data improves the structure as measured by Rfree in a zone excluding the added data, then it is hard to deny that that data are worth including. Absolutely. Much better than to believe in rules of thumb. best, Kay eab On 07/02/2015 12:52 PM, Keller, Jacob wrote: Wasn’t all of this put to bed through the implementation of CC measures? JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Robbie Joosten *Sent:* Thursday, July 02, 2015 12:46 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] paired refinement But it is not the R-free of the shell here. In paired refinement you take the R-free of the reflections outside the shell. Cheers, Robbie Sent with my Windows Phone -- *Van: *Edward A. Berry mailto:ber...@upstate.edu *Verzonden: *2-7-2015 18:43 *Aan: *CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Onderwerp: *Re: [ccp4bb] paired refinement Another criterion for cutoff, also requiring the structure to be solved, is the agreement between data and structure, e.g. Rfree or CCfree. I think it is very unlikely that you could get Rfree =.2493 in a shell which contains only noise. So I would suggest doing paired refinement to 2.2 and 2.1 A (if the data is available). On 07/01/2015 07:15 PM, Eric Karg wrote: Hi all, I have
Re: [ccp4bb] paired refinement
Comparing stats on geometry as well as R values is confounded by the problem of choosing weights. Should you hold the weights fixed or perform an individualized weight optimization at each step? More importantly, our geometry libraries are imperfect. We know from surveys that the fit of models to Engh Huber gets worst as the resolution of their X-ray data gets higher. This is because there are real (and to a good extent conformationally dependent) variations in bond angles that are brought to light by the high resolution (better than about 1.6A) data. If I add quality high resolution data I would expect the geometry stats to get worst. Dale Tronrud On 7/7/2015 11:17 AM, Shane Caldwell wrote: Chiming in late with a follow-up question: On the other hand in paired refinement, if adding the data improves the structure as measured by Rfree in a zone excluding the added data, then it is hard to deny that that data are worth including. Is it correct to think that model geometry would also be valuable at this point? If adding reflections lead to a model with more reasonable average bond angles, reduced clashes, etc., that would indicate that the added reflections have improved the refinement, no? The geometry stats should also be completely independent from the crystallographic stats. Shane Caldwell McGill University On Fri, Jul 3, 2015 at 2:56 AM, Kay Diederichs kay.diederi...@uni-konstanz.de mailto:kay.diederi...@uni-konstanz.de wrote: On Thu, 2 Jul 2015 13:25:07 -0400, Edward A. Berry ber...@upstate.edu mailto:ber...@upstate.edu wrote: My take on this- No one has been willing to specify a cutoff (and probably there is no rigorous way to mathematically define the cutoff) and say If CC* (or CCfree or whatever) is below X then it will not improve your structure, if above X then it will. the electron microscopy community uses a similar measure (FSC, Fourier Shell Correlation) as CC1/2. They follow their Gold Standard if they cut their data at FSC=0.143 . Mathematically, CC1/2=0.143 is equivalent to CC*=0.5 . So researchers of that community _do_ specify a cutoff, and by calling it Gold Standard they cast it in stone. Very helpful because probably no reviewer ever calls a Gold Standard into question. Probably depends among other things on how strong the lower resolution data is, how good the structure is without the added data. the latter point is crucial: a structure that is good can make use of higher-resolution data than a structure that is not properly refined. That is different from the situation in electron microscopy, where the phases are obtained experimentally. This is why an X-ray structure at an early stage of iterative refinement/fitting may possibly not be improved by the weak high-resolution data , and why the paired refinement should be done during the end game of refinement. But as long as CC1/2 is statistically significant it may improve a very good model even if CC*0.5 (see Bublitz et al IUCrJ 2, 409-420 (2015) for an example). To find out how close the structure to the data is, it helps to compare CCwork and CC*. An arbitrary cutoff (like CC*=0.5) is thus not sensible in all situations; it may serve as a rule of thumb though since the difference in resolution limit is not large anyway: CC*=0.5 and CC*=0.3 usually differ by (say) 0.1A only. On the other hand in paired refinement, if adding the data improves the structure as measured by Rfree in a zone excluding the added data, then it is hard to deny that that data are worth including. Absolutely. Much better than to believe in rules of thumb. best, Kay eab On 07/02/2015 12:52 PM, Keller, Jacob wrote: Wasn’t all of this put to bed through the implementation of CC measures? JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Robbie Joosten *Sent:* Thursday, July 02, 2015 12:46 PM *To:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] paired refinement But it is not the R-free of the shell here. In paired refinement you take the R-free of the reflections outside the shell. Cheers, Robbie Sent with my Windows Phone
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Re: [ccp4bb] determine NCS operators from PDB coordinates
Dear all, Thanks for your helpful emails. Here is a short summary: I followed Edward Berry's suggestions. Unfortunately I could not use any superposition to the new structure, as suggested by some, since the structures are too different. I used lsqman and mama to obtain the sperical polars for my twofold as pointed out by Ed. After this, I rotated my structure in two steps based on these angles, but using rotation matrixes in the form cos_theta -sin_theta0 sin_thetacos_theta0 0 0 1 as pointed out by Ed, to align the twofold axis in my structure to a twofold axis in the new space group. That was done in pdbset (I did not need to consider translation in my case). Thanks again for helping me! Best wishes, Tobias. On Fri, Jun 19, 2015 at 4:54 PM, Edward A. Berry ber...@upstate.edu wrote: A number of superposition programs allow to superimpose specified atoms (such as CA). Once you get the operator, comparing two different operators is not a job for a conventional superposition program, since you are superimposing a line on a line which has the extra degree of freedom- rotation about the line. If you express the operator in spherical polar coordinates, which the superposition program may provide or you can get from the matrix using ccp4 rotmat, you should be able to work out the relation between the axes. Using the Uppsala sofware factory programs: #This is superimposing parts of chains C, A, B on P, N, O (and vice versa - it's proper 2-fold) lsqman -b eof chain_mode original re m1 cbc596.pdb exp m1 C20-370 P20-370 A20-200 A250-400 B30-200 B250-400 N20-200 N250-400 O30-200 O250-400 m1 P20 C20 N20 N250 O30 O250 A20 A250 B30 B250 save m1 m1 ncsasc01.odb quit eof This prints the operator and saves it in ncsasc01.odb* - The 2010 atoms have an RMS distance of0.130 A - RMS delta B = 15.367 A2 - Corr. coeff. = 0.6601 - Rotation: -0.834671 -0.550747 -0.001321 --0.550747 0.834660 0.004400 --0.001321 0.004400 -0.89 - Translation :129.38438.428 171.594 --- Now use mama in an off-label way to convert to polar coordinates: mama overlap ncs ncsasc01.odb gives: --- - RT-OP 1 =-0.8346710 -0.5507473 -0.0013208 129.384 - -0.55074730.83466040.0044000 38.428 - -0.00132080.0044000 -0.894 171.594 - Determinant of rotation matrix 1.00 - Column-vector products (12,13,23) 0.000.000.00 - Crowther Alpha Beta Gamma 106.709 179.736 73.291 - ***Spherical polars Omega Phi Chi 90.132 -73.291 180.000*** - Direction cosines of rotation axis 0.287514 -0.957774 -0.002303 - X-PLOR polars Phi Psi Kappa *undefined* *undefined* 180.000 - Lattmann Theta+ Theta2 Theta- -180.000 179.736-146.582 - Rotation angle 180.000 - * if you use the .odb file outside of the USF software, be aware the matrix is the transpose (or more accurately it is written by columns). In ccp4 this is taken care of withthe keyword odb. On 06/19/2015 09:07 AM, Tobias Beck wrote: Dear all, I have a PDB file that contains NCS in the asymmetric unit, probably point group D3. 1.) What program is recommended for determining the symmetry operators from PDB coordinates? I found findncs, but this uses only heavy atom coordinates (I could probably use just the sulfurs from the PDB as a work around). 2.) Then I would like to compare the PDB file to a related structure. Here I would like to align the symmetry operators determined above with symmetry elements found in a different space group, for example align the twofold axis from NCS with a twofold axis in found in a particular space group. What is a good way to go about this? I am aware that NCS is used in programs as restraints during refinement, but here I am interested in obtaining the NCS symmetry operators and aligning them to symmetry elements present in a new space group. Maybe I am overlooking an obvious solution. Any help is greatly appreciated. Thanks and best wishes, Tobias. -- ___ Dr. Tobias Beck - independent group leader - RWTH Aachen University Institute of Inorganic Chemistry Landoltweg 1, office: 304N 52056 Aachen, Germany phone: +49-241-80-90057 fax: +49-241-80-99003 web: http://www.ac.rwth-aachen.de/extern/beck/ ___ -- ___ Dr. Tobias Beck - independent group leader - RWTH Aachen University Institute of Inorganic Chemistry Landoltweg 1, office: 304N 52056 Aachen, Germany phone: +49-241-80-90057 fax: +49-241-80-99003 web: http://www.ac.rwth-aachen.de/extern/beck/
Re: [ccp4bb] DNA RNA annealing problem
Hi Weifei, When I was looking at Holliday junction annealing I found that the salt concentration was important. NaCl around 300-500mM improved yield as did MgCl2 at lower (100-200mM) concentrations. So, it might be worth trying higher salt. Dave -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ChenWeiFei Sent: 03 July 2015 16:14 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA RNA annealing problem Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] DNA RNA annealing problem
Hi Weifei, so... what I do is to mix them in a 1:1ish ratio, then I check that ratio on the HPLC in case I can adjust it to 1:1. I.e. Instead of mixing 30 nmol of RNA with 30 of DNA if I want a 1:1 ratio complex, I mix 30 of RNA and 25 of DNA. I calculate their molar extinction coefficient (epsilon) and check what is the ratio between the one of RNA and the one of DNA. Out of the HPLC I calculate the area of the peaks, divide one by the other one and check whether this ratio matches the one between the epsilons. If it doesn't then you calculate how much you still need to add to achieve this 1:1 ratio. Depending on your desired final concentrartion you probably have to concentrate this RNA-DNA mixture before adding the buffer. I concentrate in viva spin colum and then add the buffer (mine is 10mM HEPES pH 8.0, 150 mM NaCl, and 2mM KCl. I anneal it at 95° for 3 minutes and then let it cool to room temperature for about 20'. After it cools down I add 20 mM MgCl2. I'm not sure this helps a lot or not... this is what I do... I don't know if it gives you any idea of what can be not working in your case. Good luck. Best, ALmudena 2015-07-03 17:14 GMT+02:00 ChenWeiFei weife...@outlook.com: Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] Post-doctoral research position at the Biozentrum, University of Basel
*Post-doctoral position: Structure and function of a large membrane protein insertase complex* A postdoctoral research position is available in the group of Sebastian Hiller at the Biozentrum, University Basel. In this project, we want to unravel the mechanism for insertion of beta-barrel membrane proteins into mitochondrial and bacterial outer membranes at atomic resolution. This process is mediated by large insertase complexes including Omp85 proteins. The project characterizes structure, function, and dynamics of such a complex using solution NMR spectroscopy and complementary techniques, in combination with suitable biochemical preparations. The project is ideally suited for a competitive researcher with experience in membrane protein biochemistry and solution NMR spectroscopy. Motivated individuals with a different background in structural biology are also strongly encouraged to apply. The Biozentrum offers an international and enthusiastic work environment with excellent infrastructure in all areas of life science. Our facilities for structural biology include 600-900 MHz Bruker NMR spectrometers, high-throughput crystallization screening, state-of-the-art cryo-electron microscopy, and a biophysics core facility. The position is available for a duration of at least two years and with a starting date within the next 6 months. Applicants should submit a CV, list of publications, a letter of motivation, and contact information for at least two reference persons. Prof. Sebastian Hiller Focal Area Structural Biology and Biophysics Biozentrum, University of Basel Klingelbergstrasse 70, 4056 Basel, Switzerland sebastian.hil...@unibas.ch http://www.biozentrum.unibas.ch/hiller/ phone: +41 61 267 20 82 fax: +41 61 267 21 09
Re: [ccp4bb] Post-doctoral research position at the Biozentrum, University of Basel
SentfrommyBlackBerry10smartphone.From: Sebastian HillerSent: Tuesday, July 7, 2015 2:47 AMTo: CCP4BB@JISCMAIL.AC.UKReply To: Sebastian HillerSubject: [ccp4bb] Post-doctoral research position at the Biozentrum, University of BaselPost-doctoral position: Structure and function of a large membrane protein insertase complex A postdoctoral research position is available in the group of Sebastian Hiller at the Biozentrum, University Basel. In this project, we want to unravel the mechanism for insertion of beta-barrel membrane proteins into mitochondrial and bacterial outer membranes at atomic resolution. This process is mediated by large insertase complexes including Omp85 proteins. The project characterizes structure, function, and dynamics of such a complex using solution NMR spectroscopy and complementary techniques, in combination with suitable biochemical preparations. The project is ideally suited for a competitive researcher with experience in membrane protein biochemistry and solution NMR spectroscopy. Motivated individuals with a different background in structural biology are also strongly encouraged to apply. The Biozentrum offers an international and enthusiastic work environment with excellent infrastructure in all areas of life science. Our facilities for structural biology include 600-900 MHz Bruker NMR spectrometers, high-throughput crystallization screening, state-of-the-art cryo-electron microscopy, and a biophysics core facility. The position is available for a duration of at least two years and with a starting date within the next 6 months. Applicants should submit a CV, list of publications, a letter of motivation, and contact information for at least two reference persons. Prof. Sebastian HillerFocal Area Structural Biology and BiophysicsBiozentrum, University of BaselKlingelbergstrasse 70, 4056 Basel, Switzerlandsebastian.hil...@unibas.chhttp://www.biozentrum.unibas.ch/hiller/phone: +41 61 267 20 82fax: +41 61 267 21 09
Re: [ccp4bb] Classifying Diverse Conformations of Many Structures of a Protein
Sounds like a perfect application for principal components analysis. Check out Bio3D: http://thegrantlab.org/bio3d/index.php. Cheers, Tristan Tristan Croll Lecturer Faculty of Health School of Biomedical Sciences Institute of Health and Biomedical Engineering Queensland University of Technology 60 Musk Ave Kelvin Grove QLD 4059 Australia +61 7 3138 6443 This email and its attachments (if any) contain confidential information intended for use by the addressee and may be privileged. We do not waive any confidentiality, privilege or copyright associated with the email or the attachments. If you are not the intended addressee, you must not use, transmit, disclose or copy the email or any attachments. If you receive this email by mistake, please notify the sender immediately and delete the original email. On 8 Jul 2015, at 11:06 am, Keller, Jacob kell...@janelia.hhmi.org wrote: Is anyone aware of a way to classify large numbers (100s) of conformationally-diverse crystal structures of a single protein (here calmodulin)? Pairwise RMSD matrixes seem possible, but may be complicated since there are two somewhat stable lobes, and the flexible linker in the middle. What I am imagining is a sort of multidimensional tree depicting the relationships in conformation space of the various structures. I remember something for this called esct or similar, but can't seem to google it. Any thoughts? Jacob *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
[ccp4bb] Classifying Diverse Conformations of Many Structures of a Protein
Is anyone aware of a way to classify large numbers (100s) of conformationally-diverse crystal structures of a single protein (here calmodulin)? Pairwise RMSD matrixes seem possible, but may be complicated since there are two somewhat stable lobes, and the flexible linker in the middle. What I am imagining is a sort of multidimensional tree depicting the relationships in conformation space of the various structures. I remember something for this called esct or similar, but can't seem to google it. Any thoughts? Jacob *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
[ccp4bb] [Job] Postdoctoral Position in Structural Biology at Mayo Clinic, Rochester, USA
Postdoctoral Position in Structural Biology at Mayo Clinic, Rochester, USA A Postdoctoral position is available to a highly motivated individual trained in structural biology. The project will involve the structural and dynamic characterization of large protein complexes broadly involved in chromatin remodeling and the DNA damage response. Funding for this position is secured for up to 5 years. Please email a CV and a letter of motivation to Georges Mer: mer.geor...@mayo.edu A Postdoctoral Research Fellow at Mayo Clinic is a temporary position supported by a competitive salary and benefits package. Qualified individuals will demonstrate potential for research as evidenced by their training and peer-reviewed publications.
