Re: [ccp4bb] artificial tetramer
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6G0aUxlJ7aRr7hoRAlMyAJ9ekKa8oatBgpiNsumGCzJtG1Gu9QCgq5Q7 ZDe36RL5FfSU1usr2p7AZxw= =QHqV -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6G0aUxlJ7aRr7hoRAlMyAJ9ekKa8oatBgpiNsumGCzJtG1Gu9QCgq5Q7 ZDe36RL5FfSU1usr2p7AZxw= =QHqV -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
This sounds as though you will create a 4-helical rod? There are lots of 4-helical bundles but they arenot completely rod like - there is a twist in most I have seen. Maybe you should do secondary structure matching to a suitable 4-helicl bundle, match your helix in turn to each of the bundle helices. Eleanor On 12/14/2011 02:13 PM, Fred wrote: Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6G0aUxlJ7aRr7hoRAlMyAJ9ekKa8oatBgpiNsumGCzJtG1Gu9QCgq5Q7 ZDe36RL5FfSU1usr2p7AZxw= =QHqV -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
Dear Fred, In this case, I would take one monomer and edit the CRYST1 card of the pdb to have a sufficiently large tetragonal unit cell. Next I would manually align the helix of one monomer with the fourfold axis, e.g. the Z-axis. If you display the unit cell, you will get e.g. the 0,Z,0 axis to guide you. Then I would manually translate the monomer such that it forms a nice tetramer with its symmetry mates and would write out these symmetry mates. Once you have one good tetramer, you can (or have somebody) write a script to superimpose all your other monomers onto this template. My two cents,... Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Fred Sent: Wednesday, December 14, 2011 3:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] artificial tetramer Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6G0aUxlJ7aRr7hoRAlMyAJ9ekKa8oatBgpiNsumGCzJtG1Gu9QCgq5Q7 ZDe36RL5FfSU1usr2p7AZxw= =QHqV -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Fred, as far as I understand your problem I am not aware of existing programs to carry out the tasks. To automatise the process I would write a program myself which takes the PDB-file and the reside numbers which make that helix as input and: 1) determine the axis of that straight helix (probably by determining the moments of intertia, see e.g. Gruene, T. Sheldrick, G. M. (2011). Acta Cryst. A67, 1-8, section 2.2.1 with x_o the centre of mass. 2) copy the monomer by 3 rotations of 90 degrees about this axis to get the tetramer. 3) move each of the four monomers stepwise perpendicularly to the rotation axis until no more clashes are there (mask the volume of one monomer and check each atom/atom volume whether it falls into that volume. Because of the 4-fold rotation axis you only need to mask one monomer and check against its next and its opposite neighbour). The tricky bit w.r.t automatisation would be to determine the helix axis, unless you already have a list of composing residues for each PDB-file. That's probably nothing I would finish in a week's time, though... Tim On 12/14/2011 03:13 PM, Fred wrote: Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6LusUxlJ7aRr7hoRAk5QAJ0ewv3ZjLRSU9fF1c2xBDDypcLX6ACg5+2P X01grlPsYcSeWZbQWIVtXD0= =Fse5 -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
An old old trick for getting a straight helix along a crystal axis using the GUI. Run the Amore TABLING step - it is part of the GUI with the helix as your model. (See molecular replacement - amore suite.) Amore calculates the ppl axes and will align it along the Z axis .. Thank you Jorge.. Eleanor On 12/14/2011 03:07 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Fred, as far as I understand your problem I am not aware of existing programs to carry out the tasks. To automatise the process I would write a program myself which takes the PDB-file and the reside numbers which make that helix as input and: 1) determine the axis of that straight helix (probably by determining the moments of intertia, see e.g. Gruene, T. Sheldrick, G. M. (2011). Acta Cryst. A67, 1-8, section 2.2.1 with x_o the centre of mass. 2) copy the monomer by 3 rotations of 90 degrees about this axis to get the tetramer. 3) move each of the four monomers stepwise perpendicularly to the rotation axis until no more clashes are there (mask the volume of one monomer and check each atom/atom volume whether it falls into that volume. Because of the 4-fold rotation axis you only need to mask one monomer and check against its next and its opposite neighbour). The tricky bit w.r.t automatisation would be to determine the helix axis, unless you already have a list of composing residues for each PDB-file. That's probably nothing I would finish in a week's time, though... Tim On 12/14/2011 03:13 PM, Fred wrote: Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6LusUxlJ7aRr7hoRAk5QAJ0ewv3ZjLRSU9fF1c2xBDDypcLX6ACg5+2P X01grlPsYcSeWZbQWIVtXD0= =Fse5 -END PGP SIGNATURE-
Re: [ccp4bb] artificial tetramer
I've done this sort of thing (but different) using the Clipper libraries in C++. I found it easier to write short programs to do specific tasks rather than try to write generic all-purpose tools Phil On 14 Dec 2011, at 15:07, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Fred, as far as I understand your problem I am not aware of existing programs to carry out the tasks. To automatise the process I would write a program myself which takes the PDB-file and the reside numbers which make that helix as input and: 1) determine the axis of that straight helix (probably by determining the moments of intertia, see e.g. Gruene, T. Sheldrick, G. M. (2011). Acta Cryst. A67, 1-8, section 2.2.1 with x_o the centre of mass. 2) copy the monomer by 3 rotations of 90 degrees about this axis to get the tetramer. 3) move each of the four monomers stepwise perpendicularly to the rotation axis until no more clashes are there (mask the volume of one monomer and check each atom/atom volume whether it falls into that volume. Because of the 4-fold rotation axis you only need to mask one monomer and check against its next and its opposite neighbour). The tricky bit w.r.t automatisation would be to determine the helix axis, unless you already have a list of composing residues for each PDB-file. That's probably nothing I would finish in a week's time, though... Tim On 12/14/2011 03:13 PM, Fred wrote: Hi Tim, Thanks for your replay. All pdb monomers have the same primary sequence and a perfect matching long helix, which I have used to superpose the coordinates. Such helix is almost straight so that, the idea would be to create a vector along the helix main axis, shift this axis to a some distance (perhaps minimizing clashes) and apply 4-fold rotation. A second step would be to take these into pdbset to make things in batch mode. It sounds simple, but don't know the easiest way/programs to do that. I can do just the basics in Coot. I remember that Xfit had some options to trace vectors inside a cell and give it rotation properties. However, Xfit seems to be frozen and integration with pdbset would be painful. Regards, Fred Em 14-12-2011 07:32, Tim Gruene escreveu: Hi Fred, this sentence of yours, All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. probably hides the information which would be necessary for a reasonable answer to your question. If you still are stuck, you might post again with a more detailed description of what you mean. Cheers, Tim On 12/13/2011 07:28 PM, Fred wrote: Dear CCP4bb list, Thank you very much all of you who have answered my post. I'm really sorry if I was unclear. Such operation is so unusual that I could be able to express myself appropriately. From quick reading some replies (James Stroud and Guillaume Ponchel), it seems is possible do build artificial tetramers with Coot. Several problems have been raised like clashes, unusual interfaces and so on. A second step would be to take Coot's rotation and translation matrix and apply it to all pdb's in batch mode with pdbset. All pdb's are superposed by a common sequence region, which also will be part of the tetramer interface. I'll try to make things work. Once more, sorry for any inconvenience and thank you very much. Kind regards, Fred -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO6LusUxlJ7aRr7hoRAk5QAJ0ewv3ZjLRSU9fF1c2xBDDypcLX6ACg5+2P X01grlPsYcSeWZbQWIVtXD0= =Fse5 -END PGP SIGNATURE-
Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
Jürgen, Have you checked a simple selfrotation function in your currently favored space group ? Yes, both selfrotation function and patterson map do not look suspicious in I422. Do you have sufficient data collected to start out in P1 or C2 ? Then I would start there and systematically look at selfrotation functions in those space groups. Also check the native Patterson for translational NCS. I got ~220 degrees before the radiation damage became significant, so there should be enough data and I will look into it. 4 A is not great for stable refinement of cell parameters, which program did you use and which parameters did you fix? HKL2000 without fixing parameters. mosflm can't hold on the lattice and I haven't tried d*trek, yet. Did you use main.ncs=true in the SA approach ? yes Pointless or xtriage ? xtriage Why does it take a year to grow those crystals ? Well, other crystals don't diffract and the protein is quite stable in solution. That's how it is, I guess? Out of curiosity, how did you collect on this crystal without overlapping reflections ? I got away with 1 second exposure and 0.75 degree oscillation at 650mm detector distance. there are some predicted overlaps but they are in those regions that are empty due to anisotropy. Our cryo condition gives well separated small spots of nice, round shape, mosaicity is ~0.6. The pattern itself looks great, although the beamstop shadow is quite big on the frames. Thank You, Stefan
Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
Give XDS a try with your data or d*trek with 3d profile fitting. Did you try iMosflm or the old Mosflm ? If old, then POSTREF WIDTH 15 might help and POSTREF FIX BEAM You saw the nice packing from Tjaard, if all your molecules have contact with each other than that's fine I was just concerned about molecules in space without contact, then you are missing something. Still reprocessing the data (sorry Wladek) might squeeze out a bit more of your existing data. In particular if you use NCS averaging with those weak highres reflections you might get better side chain density. Jürgen On Dec 14, 2011, at 11:41 AM, Stefan Gajewski wrote: Jürgen, Have you checked a simple selfrotation function in your currently favored space group ? Yes, both selfrotation function and patterson map do not look suspicious in I422. Do you have sufficient data collected to start out in P1 or C2 ? Then I would start there and systematically look at selfrotation functions in those space groups. Also check the native Patterson for translational NCS. I got ~220 degrees before the radiation damage became significant, so there should be enough data and I will look into it. 4 A is not great for stable refinement of cell parameters, which program did you use and which parameters did you fix? HKL2000 without fixing parameters. mosflm can't hold on the lattice and I haven't tried d*trek, yet. Did you use main.ncs=true in the SA approach ? yes Pointless or xtriage ? xtriage Why does it take a year to grow those crystals ? Well, other crystals don't diffract and the protein is quite stable in solution. That's how it is, I guess? Out of curiosity, how did you collect on this crystal without overlapping reflections ? I got away with 1 second exposure and 0.75 degree oscillation at 650mm detector distance. there are some predicted overlaps but they are in those regions that are empty due to anisotropy. Our cryo condition gives well separated small spots of nice, round shape, mosaicity is ~0.6. The pattern itself looks great, although the beamstop shadow is quite big on the frames. Thank You, Stefan .. 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] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
How about plotting the solvent content along with resolution limits of the structures? From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel Afonine Sent: Wednesday, December 14, 2011 12:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning Hi Stefan, 1) just out of curiosity I wrote a tiny script using CCTBX that estimates solvent content via bulk-solvent mask, and quickly run this script for all PDB structures for which I could re-calculate the R-work within 5% from published value. Also, this script extracted the solvent content values reported in PDB file header. Here is what I get: Histogram of solvent contents (estimated via mask): Solvent content Number of structures 5.980 - 14.482 : 11 14.482 - 22.984 : 109 22.984 - 31.486 : 396 31.486 - 39.988 : 3590 39.988 - 48.490 : 11442 48.490 - 56.992 : 11707 56.992 - 65.494 : 6524 65.494 - 73.996 : 2561 73.996 - 82.498 : 510 82.498 - 91.000 : 19 Histogram of solvent contents (extracted from REMARK records): Solvent content Number of structures 6.000 - 14.300 : 91 14.300 - 22.600 : 550 22.600 - 30.900 : 2046 30.900 - 39.200 : 6487 39.200 - 47.500 : 9566 47.500 - 55.800 : 9050 55.800 - 64.100 : 5853 64.100 - 72.400 : 2420 72.400 - 80.700 : 720 80.700 - 89.000 : 86 So, your 78% is not that uncommon although it is at the high(ish) end. 2) Does Xtriage suggest twinning? If so what happens if you refine with the twin law? 3) Make sure you look at both, 2mFo-DFc with and without missing Fobs filled with DFc (depending on completeness of your data that may make a big difference). Pavel On Tue, Dec 13, 2011 at 8:47 PM, Stefan Gajewski sgajew...@gmail.commailto:sgajew...@gmail.com wrote: I am looking at a highly unusual crystal lattice right now and can't figure out what is going on, so I decided to ask the experts. I recently got data on a oligomeric protein with many highly correlated NCS units (4.0A resolution, linear R-sym is 0.16-0.21 in I4, I422, F222, C2 and 0.12 in P1) with severe anisotropic diffraction (according to diffraction anisotropy server, the F/sigma drops below 3 at a=6.1 b=6.1 c=never, suggested isotropic B-sharpening -125A^2) This lattice has a problem. The apparent unit cell is rather huge (roughly 180 180 620 / 90 90 90) The unit cell dimensions are almost perfectly I4 and the presence of systematic absent reflections 50 I/s in I41 and I4122 suggest no screw axis. I used a very closely related structure solved at 4.2A as molecular replacement model and got a solution from the anisotropy corrected data in I422 space group with two oligomers in the asymmetric unit cell. Confidence of the MR solution is quite high since (a)the MR replacement put one model one NCS raster off the true position resulting in a clash with the second one in an empty region of the map and additional electron density on the other side which corresponds perfectly to the wrongly positioned monomer, and (b) after rotating the model in the right position I could refine the structure to R-work=0.31. R-free=0.35 in one run of rigid body refinement followed by NCS restrained simulated annealing refinement (phenix.refine), which is in my opinion really good at such an early stage of refinement given the low overall resolution and even lower completeness of strong reflections in a and b due to high anisotropy (observables to atoms ratio is about 3:1) . I can even see clear density for some of the bulky sidechains which were not included in the model. Now here is the baffling thing. The unit cell is almost empty with an apparent solvent content of 78%. The molecules cluster around the c-axis and at the origin with an empty gap in a and b of at least 15A and up to 165A(!) in the longest dimension. There is no sign of electron density that would indicate a missing protein in that region and ~98% of my model is already accounted for by the density in the 2Fo-Fc map, making a contact of disordered protein regions across the ASUs unlikely. In fact, the protein density is well defined at the closest gap and no mainchain atom is unaccounted for in that region. The oligomer has a magnitude of ~105A x 70A. I heavily doubt that a crystal lattice with such little contacts and holes as huge as these can exist and therefore think that: (a) the R-factors are misleading me to think the solution is correct and complete (b) I must have been doing something really wrong Since proteins from this family have a well established history of producing twinned crystals I had a look at that possibility. Analyzing the anisotropy corrected I4 data for twinning (Padilla Yeates method) revealed a 2-fold twin law with a twin fraction of 0.42 which would make
Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning
Hi Debasish, On Wed, Dec 14, 2011 at 9:23 AM, Debasish Chattopadhyay debas...@uab.eduwrote: How about plotting the solvent content along with resolution limits of the structures? here it is: Resolution Number of Solvent % (mean/min/max) Solvent % (mean/min/max) range structures from remarks from mask 0.0-1.0 148 (37.3 9.9 61.7) (30.7 9.0 55.0) 1.0-1.5 2934 (44.6 6.0 75.0) (37.4 9.0 77.0) 1.5-2.0 14270 (48.7 16.4 81.1) (43.6 8.0 77.0) 2.0-2.5 11432 (52.6 16.9 84.0) (50.8 7.0 83.0) 2.5-3.0 5467 (57.0 27.0 86.0) (57.5 25.0 88.0) 3.0-3.5 1140 (61.3 20.0 89.7) (62.6 30.0 89.0) 3.5-4.0 151 (65.9 35.0 85.0) (67.1 45.0 88.0) 4.0-9.027 (66.2 43.0 84.4) (67.4 50.0 81.0) Pavel
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
On 13 December 2011 17:59, James Holton jmhol...@lbl.gov wrote: A small but potentially important correction: FC_ALL PHIC_ALL from REFMAC are indeed the calculated structure factor of the coordinates+bulk_solvent, but AFTER multiplying by the likelihood coefficient D (as in 2*m*Fo-D*Fc ). So, if you subtract ( FC_ALL PHIC_ALL ) from ( FC PHIC ) you will NOT get the bulk solvent contribution alone. AFAIK there is no way to obtain just the bulk solvent contribution from REFMAC. James, I agree completely! But I would venture to go further and say that the FC/PHIC values really have no business being in the output MTZ file in the first place, so if they weren't there then the question of subtracting them would never arise. They are the result of intermediate calculations, the kind of things I print out when I'm debugging a program to aid in checking the logic. The FC_ALL/PHIC_ALL values represent the final definitive result of the refinement, so in all applications where Fcalc is required (e.g. density correlation stats) DFc/phi(DFc) should always be used - and why would one want to omit part of the model anyway (unless maybe for an omit map - but that doesn't seem to be relevant here)? Fc/phic is the transform of the refined atomic model parameters as output in XYZOUT which essentially is just a snapshot of the model. DFc/phi(DFc) represents the transform of an ensemble average of a distribution of models generated by the random co-ordinate (and other parameter) errors, and of course everyone knows that X-ray diffraction measures the ensemble average, not a shapshot. Also we know that (2mFo-DFc)/phic (or mFo/phic if centric) is the best estimate of the true phased F. The best estimate of the difference Ftrue-Fmodel is the difference coefficient 2(mFo-DFc)/phic (or mFo-DFc)/phic if centric). So the best estimate of Fmodel is clearly (Ftrue - (Ftrue - Fmodel))/phic = (2mFo - DFc - 2(mFo - DFc))/phic = DFc/phic (and the same result for centric). Cheers -- Ian
[ccp4bb] off-topic: Phoenix robot problems
Dear CCP4ers, We are currently fighting with our crystallization robot and would be happy to find out if anyone else has experienced similar problems - and hopefully found a solution. To make a long and extremely frustrating story with our robot short: We are running an ArtRobbins 4-Channel Phoenix in our xtal-facility. It is equipped with the new nano-dispensers and TC-needles which are probably also present in the Cobra or Gryphon instruments. Our problem is that almost every two days we have serious trouble with the nano-dispensing needles, which either become leaky or completely clogged. We do not even heavily use our Phoenix or use membrane proteins in detergents - in average we are dispensing less than 5-10plates of usually nice behaving proteins per day. The same proteins dispensed with the old needle systems (in the neighboring MPI facility) are not causing any problems at all. I am not really sure how to avoid these problems or how to deal with it. We do almost everything with our samples to remove aggregates (sample centrifuged 10min@13k rpm, supernatant filtered again through filter units). I am happy to receive any comments or hints regarding the Phoenix (or Cobra/Gryphon if the nanodispensers are the same type) or any additional comment e.g. on how you prepare samples in your lab. I would be grateful if anyone with ideas or suggestions could write to me off-list, I will post a summary if we can solve the problems. Thanks and regards from munich, Gregor --- Dr. Gregor Witte Genecenter, University of Munich (LMU) Feodor-Lynen-Str. 25 D-81377 Munich mail: wi...@genzentrum.lmu.de
[ccp4bb] Hydrophobic interactions
Hi Everyone I have to calculate hydrophobic interactions in pdb files. Is any server available for this type of calculation? Thanks in advance Amit
[ccp4bb] Mg or water?
Hi every, I'm working with 2 crystal forms of a protein from 2 different crystallization conditions. Condition 1 has 100mM MgCl2. Condition 2 doesn't. Both are ~2.9 angstrom. The 2 structures are virtually identical except in condition1, there is a clear positive density surrounded by a Glu side chain carboxyl and a couple of main carboxyl groups. (Again, condition 2 doesn't have this density). My initial thought is that a Mg atom is incorporated and it fits well. But the problem is we can not role out the possibility of a water molecule. Refining with Mg gives a b-factor of 42 (about average for the whole protein). The b-factor is 21 when refining with a water. Both cases there is no positive/negative density at contour=2.0. Based on the current data, is there any other role we can apply to see how likely it is a Mg or water. Or anomalous scattering is the only way? Thanks for your suggestions. Best, Gao
[ccp4bb] Pore Dimension Convention
Dear Crystallographers, is there a convention for denoting/measuring pore sizes in protein structures? Maybe inter-atom distances minus van der Waals radii? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Pore Dimension Convention
Hi Jacob, There are a number of programs that can calculate the radius of a pore. The one that comes to mind is called HOLE, and it can make a nice plot of the y-coordinate along the pore vs. pore radius. I don't recall exactly how this calculation is done, I think it is somehow related to the SASA (some sort of spherical probe type of thing), but I'm sure you can have a look at the documentation for more details. A quick search also shows that there are apparently a number of tools out there that will do similar analyses. See this web page for a summary: http://www.caver.cz/index.php?sid=133 Good luck, Mike - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, December 14, 2011 3:14:33 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Pore Dimension Convention Dear Crystallographers, is there a convention for denoting/measuring pore sizes in protein structures? Maybe inter-atom distances minus van der Waals radii? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Hydrophobic interactions
google on pisa pdbe -- Eugene From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Luthra,Amit [alut...@uchc.edu] Sent: Wednesday, December 14, 2011 8:34 PM To: ccp4bb Subject: [ccp4bb] Hydrophobic interactions Hi Everyone I have to calculate hydrophobic interactions in pdb files. Is any server available for this type of calculation? Thanks in advance Amit
