[ccp4bb] AW: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site
Dear Armando, Is 1.9Å really the diffraction limit of your crystals, or do they diffract further and is 1.9Å just a convenient resolution cutoff? In the latter case you might be looking at truncation effects. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Armando Albert Gesendet: Freitag, 18. Juli 2014 18:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site Dear all, I am screening a small library of ligands against my protein crystals. Following a soaking with different ligands, I collect datasets to 1.9A resolution and refine them against an empty model without any problem. What is the meaning of a rather large negative electron density in the Fo-Fc map at the binding site?. Could it be related to an incorrect bulk solvent model? Thank you in advance Armando
[ccp4bb] Protein Crystallography challenges Standard Model precision
I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits.. Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] AW: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site
Well yes, The bulk solvent model can distort the density, especially if the ligand is large. But it usually isnt too serious - try doing SIMPLE scaling and see if that has any effect on the appearance of the density Eleanor On 22 July 2014 10:16, herman.schreu...@sanofi.com wrote: Dear Armando, Is 1.9Å really the diffraction limit of your crystals, or do they diffract further and is 1.9Å just a convenient resolution cutoff? In the latter case you might be looking at truncation effects. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Armando Albert Gesendet: Freitag, 18. Juli 2014 18:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site Dear all, I am screening a small library of ligands against my protein crystals. Following a soaking with different ligands, I collect datasets to 1.9A resolution and refine them against an empty model without any problem. What is the meaning of a rather large negative electron density in the Fo-Fc map at the binding site?. Could it be related to an incorrect bulk solvent model? Thank you in advance Armando
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Bernhard, A look at the methods section might give you a clue. Neither XDS nor XSCALE create mmCIF - files (you are talking about mmCIF, not CIF - subtle, but annoying difference), so that the choice is limited. I guess some programmer (rather than a scientist ;-) )used a simple printf commmand for a double precision number so the junk is left over from the memory region or other noise common to conversions. XDS actually prints error estimates for the cell dimensions in CORRECT.LP which could be added to the mmCIF file - a cif (sic!) file, I believe, requires those, by the way and checkCIF would complain about their absence. Cheers, Tim On 07/22/2014 01:01 PM, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits.. - Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ --- - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTzk52UxlJ7aRr7hoRAul8AKCHFz/DAoqR7s0fGUp79xx2QlrfCQCeIiiy KXSurhgaQjhguKr9L0/zyVk= =vqGC -END PGP SIGNATURE-
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
I took a look at both the PDB and CIF headers for the coordinates for 4C69 and they have normal looking numbers with three digits following the decimal point. According to the coordinate file header this entry was processed by PDBE. It would be interesting to hear from the PDBE staff if the structure factor file arrived with those numbers or if they were introduced during processing. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Tue, 22 Jul 2014, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta 99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits?. Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
[ccp4bb] Off topic: Enzyme mechanism
Hi all, Does anybody know of a case where the same enzyme from different species, with reasonably invariant active site residues, perform identical biochemical function but have significantly different substrate binding modes/mechanism of action on the same substrate? Thanks Komal
[ccp4bb] off-topic: protein ligation and phospho-tyrosine affinity chromatography
Dear all, We are planning to ligate a phospho-tyrosine containing 16aa/33aa peptide onto a 70 kDa protein and subsequently use it for crystallography studies. We will be using the Intein-fusion protein system (IMPACT kit from NEB) to accomplish the ligation. The idea is to separate the ligated protein from the non-ligated using phospho-tyrosine affinity chromatography. We have been doing a lot of literature survey for both the ligation and the purification protocols but there is no established method for achieving crystallization grade ligated products. We would really appreciate to receive inputs and be imparted a few tricks for both the ligation and the purification methods. Many thanks, Vasan
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
Error estimates for the unit cell dimensions in macromolecular crystallography belong to atypical category of uncertainty estimates. Random error contribution in most cases is below 0.001A, so it can be neglected. Wavelength calibration error can be also made very small; however, I do not know how big it is in practice. Goniostat wobble error is taken into account in Scalepack refinement. Crystal-to-detector distance is not used in postrefinement/global refinement. Due to the measurement error being very small, even small variations in unit cell parameters can be detected within cryocooled crystals. These variations almost always are _orders_of_magnitude_larger_ than measurement uncertainty. Current practise is not to investigate the magnitude of the changes in the unit cell parameters, but when beam smaller than crystal is used, observing variations as large as 1A is not unusual. The main question is: what the unit cell uncertainty means? For most samples I could defend to use values: 0.001A, 0.01A, 0.1A and 1A as reasonable, depending on particular point of view. Without defining what the unit cell uncertainty means, publishing its values is pointless. Zbyszek Otwinowski -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Bernhard, A look at the methods section might give you a clue. Neither XDS nor XSCALE create mmCIF - files (you are talking about mmCIF, not CIF - subtle, but annoying difference), so that the choice is limited. I guess some programmer (rather than a scientist ;-) )used a simple printf commmand for a double precision number so the junk is left over from the memory region or other noise common to conversions. XDS actually prints error estimates for the cell dimensions in CORRECT.LP which could be added to the mmCIF file - a cif (sic!) file, I believe, requires those, by the way and checkCIF would complain about their absence. Cheers, Tim On 07/22/2014 01:01 PM, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits.. - Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ --- - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTzk52UxlJ7aRr7hoRAul8AKCHFz/DAoqR7s0fGUp79xx2QlrfCQCeIiiy KXSurhgaQjhguKr9L0/zyVk= =vqGC -END PGP SIGNATURE- Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
Dear Zbyszek, when you optimise a set of parameters against a set of data, I guess you can also provide their errors. If I understand correctly, this comes with least-squares-routines. I only pointed out that cell errors are listed in the XDS output (provided you refine them, of course). I am sure those errors are well defined. Best wishes, Tim On 07/22/2014 06:53 PM, Zbyszek Otwinowski wrote: Error estimates for the unit cell dimensions in macromolecular crystallography belong to atypical category of uncertainty estimates. Random error contribution in most cases is below 0.001A, so it can be neglected. Wavelength calibration error can be also made very small; however, I do not know how big it is in practice. Goniostat wobble error is taken into account in Scalepack refinement. Crystal-to-detector distance is not used in postrefinement/global refinement. Due to the measurement error being very small, even small variations in unit cell parameters can be detected within cryocooled crystals. These variations almost always are _orders_of_magnitude_larger_ than measurement uncertainty. Current practise is not to investigate the magnitude of the changes in the unit cell parameters, but when beam smaller than crystal is used, observing variations as large as 1A is not unusual. The main question is: what the unit cell uncertainty means? For most samples I could defend to use values: 0.001A, 0.01A, 0.1A and 1A as reasonable, depending on particular point of view. Without defining what the unit cell uncertainty means, publishing its values is pointless. Zbyszek Otwinowski Hi Bernhard, A look at the methods section might give you a clue. Neither XDS nor XSCALE create mmCIF - files (you are talking about mmCIF, not CIF - subtle, but annoying difference), so that the choice is limited. I guess some programmer (rather than a scientist ;-) )used a simple printf commmand for a double precision number so the junk is left over from the memory region or other noise common to conversions. XDS actually prints error estimates for the cell dimensions in CORRECT.LP which could be added to the mmCIF file - a cif (sic!) file, I believe, requires those, by the way and checkCIF would complain about their absence. Cheers, Tim On 07/22/2014 01:01 PM, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits.. Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ --- Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] Part-time Work for Graduate Students
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[ccp4bb] Off-Topic Q / X Ray FLuoresence Scan Vs XANES
Doing Fluroscence scan for protein crystal at Cu wavelength prior MAD or SAD experiment shows usually fig like this https://www.dropbox.com/s/800463dlcmws6d4/fig.png 1- Fig A should represent X ray fluorescence scan. is it the same as X ray absorption scan. what does count represent in fig A (is it equal to the intensity of the emission). 2- i am wondering whether F double prime in Fig B is just a fit for counts in Fig A, it looks the same. could F or counts be converted to I/I0 or F/I0 to replot the data against these value as in XANES. in other word, Is X ray fluorescence scan in fig A the same as X-ray absorption near-edge structure (XANES)? could XANES be collected at the same beamline where diffraction data are collected for crystal ? 3- could data from Fluorescence scan in Fig A or B used to determine the oxidation or coordination number for the metal say copper for example in protein? thank YOU regards T. DawoD
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
The least-square procedure for unit cell parameter refinement provides very precise estimates of uncertainty. Why they are so precise? Because we use many thousands of unmerged reflections to determine the precision 1 to 6 parameters (unit cell parameters). However, although error propagation through the least squares provides precision of about 0.001 A, or better in some cases, this is only precision not accuracy, and the precision is calculated typically with respect to the unit cell parameters averaged across the exposed volume of a crystal. In practice, the range of unit cell parameters within a crystal can be quite broad, and when we consider accuracy it is not clear, which unit cell parameters should be a reference point. Typically, the distribution of unit cell parameters in a crystal will not follow Gaussian distribution. Therefore, the accuracy of unit cell parameters determination is not well defined, even when we know the experimental conditions very well and propagate experimental uncertainties correctly. Variability of unit cell parameters can be quite high for data sets from different samples. However, description of this variability is typically not related to the very high precision of determination of unit cell parameters for an individual sample. Zbyszek On 07/22/2014 12:33 PM, Tim Gruene wrote: Dear Zbyszek, when you optimise a set of parameters against a set of data, I guess you can also provide their errors. If I understand correctly, this comes with least-squares-routines. I only pointed out that cell errors are listed in the XDS output (provided you refine them, of course). I am sure those errors are well defined. Best wishes, Tim On 07/22/2014 06:53 PM, Zbyszek Otwinowski wrote: Error estimates for the unit cell dimensions in macromolecular crystallography belong to atypical category of uncertainty estimates. Random error contribution in most cases is below 0.001A, so it can be neglected. Wavelength calibration error can be also made very small; however, I do not know how big it is in practice. Goniostat wobble error is taken into account in Scalepack refinement. Crystal-to-detector distance is not used in postrefinement/global refinement. Due to the measurement error being very small, even small variations in unit cell parameters can be detected within cryocooled crystals. These variations almost always are _orders_of_magnitude_larger_ than measurement uncertainty. Current practise is not to investigate the magnitude of the changes in the unit cell parameters, but when beam smaller than crystal is used, observing variations as large as 1A is not unusual. The main question is: what the unit cell uncertainty means? For most samples I could defend to use values: 0.001A, 0.01A, 0.1A and 1A as reasonable, depending on particular point of view. Without defining what the unit cell uncertainty means, publishing its values is pointless. Zbyszek Otwinowski Hi Bernhard, A look at the methods section might give you a clue. Neither XDS nor XSCALE create mmCIF - files (you are talking about mmCIF, not CIF - subtle, but annoying difference), so that the choice is limited. I guess some programmer (rather than a scientist ;-) )used a simple printf commmand for a double precision number so the junk is left over from the memory region or other noise common to conversions. XDS actually prints error estimates for the cell dimensions in CORRECT.LP which could be added to the mmCIF file - a cif (sic!) file, I believe, requires those, by the way and checkCIF would complain about their absence. Cheers, Tim On 07/22/2014 01:01 PM, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits.. Bernhard Rupp k.-k. Hofkristallamt Crystallographiae Vindicis Militum Ordo b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ --- Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] Protein Crystallography challenges Standard Model precision
Shouldn't the cell dimensions be identical in the coordinate file and in the structure factor file? In this case they are not. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Tue, 22 Jul 2014, Zbyszek Otwinowski wrote: The least-square procedure for unit cell parameter refinement provides very precise estimates of uncertainty. Why they are so precise? Because we use many thousands of unmerged reflections to determine the precision 1 to 6 parameters (unit cell parameters). However, although error propagation through the least squares provides precision of about 0.001 A, or better in some cases, this is only precision not accuracy, and the precision is calculated typically with respect to the unit cell parameters averaged across the exposed volume of a crystal. In practice, the range of unit cell parameters within a crystal can be quite broad, and when we consider accuracy it is not clear, which unit cell parameters should be a reference point. Typically, the distribution of unit cell parameters in a crystal will not follow Gaussian distribution. Therefore, the accuracy of unit cell parameters determination is not well defined, even when we know the experimental conditions very well and propagate experimental uncertainties correctly. Variability of unit cell parameters can be quite high for data sets from different samples. However, description of this variability is typically not related to the very high precision of determination of unit cell parameters for an individual sample. Zbyszek On 07/22/2014 12:33 PM, Tim Gruene wrote: Dear Zbyszek, when you optimise a set of parameters against a set of data, I guess you can also provide their errors. If I understand correctly, this comes with least-squares-routines. I only pointed out that cell errors are listed in the XDS output (provided you refine them, of course). I am sure those errors are well defined. Best wishes, Tim On 07/22/2014 06:53 PM, Zbyszek Otwinowski wrote: Error estimates for the unit cell dimensions in macromolecular crystallography belong to atypical category of uncertainty estimates. Random error contribution in most cases is below 0.001A, so it can be neglected. Wavelength calibration error can be also made very small; however, I do not know how big it is in practice. Goniostat wobble error is taken into account in Scalepack refinement. Crystal-to-detector distance is not used in postrefinement/global refinement. Due to the measurement error being very small, even small variations in unit cell parameters can be detected within cryocooled crystals. These variations almost always are _orders_of_magnitude_larger_ than measurement uncertainty. Current practise is not to investigate the magnitude of the changes in the unit cell parameters, but when beam smaller than crystal is used, observing variations as large as 1A is not unusual. The main question is: what the unit cell uncertainty means? For most samples I could defend to use values: 0.001A, 0.01A, 0.1A and 1A as reasonable, depending on particular point of view. Without defining what the unit cell uncertainty means, publishing its values is pointless. Zbyszek Otwinowski Hi Bernhard, A look at the methods section might give you a clue. Neither XDS nor XSCALE create mmCIF - files (you are talking about mmCIF, not CIF - subtle, but annoying difference), so that the choice is limited. I guess some programmer (rather than a scientist ;-) )used a simple printf commmand for a double precision number so the junk is left over from the memory region or other noise common to conversions. XDS actually prints error estimates for the cell dimensions in CORRECT.LP which could be added to the mmCIF file - a cif (sic!) file, I believe, requires those, by the way and checkCIF would complain about their absence. Cheers, Tim On 07/22/2014 01:01 PM, Bernhard Rupp wrote: I am just morbidly curious what program(s) deliver/mutilate/divine these cell constants in recent cif files: data_r4c69sf # _audit.revision_id 1_0 _audit.creation_date ? _audit.update_record 'Initial release' # _cell.entry_id 4c69 _cell.length_a 100.152000427 _cell.length_b 58.3689994812 _cell.length_c 66.5449981689 _cell.angle_alpha 90.0 _cell.angle_beta99.2519989014 _cell.angle_gamma 90.0 # Maybe a little plausibility check during cif generation might be ok Best, BR PS: btw, 10^-20 meters (10^5 time smaller than a proton) in fact seriously challenges the Standard Model limits..
