Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?
Dear Francois, my starting point would be 'man areaimol' which also contains the references REFERENCES 1. B.Lee and F.M.Richards, J.Mol.Biol., 55, 379-400 (1971) 2. E.B.Saff and A.B.J.Kuijlaars, The Mathematical Intelligencer, 19, 5-11 (1997) http://www.math.vanderbilt.edu/~esaff/texts/161.pdf Cheers, Tim On Thu, Jan 13, 2011 at 03:00:54PM +0900, Francois Berenger wrote: Hello, Does someone know some good articles on this particular topic? I'd like to implement the thing myself, however if there is a good software doing the job (with readable source code), I might use and cite it. Best regards, Francois. -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] Phase Separation
Dear all, I'm trying to crystallize a small, soluble part of a protein (~15kDa, 152AA). I did some standard screens (Crystal Screen I II + Index screen) with a protein concentration of 25 or 45mg/mL in an 1:1 (0.75µL)96 well set up. In most of the conditions I got phase separation (mostly PEG conditions)! Precipitation was formed in conditions with salt. I did not have phase separation with the control (buffer only, see below). For so far I know my protein was soluble up to a concentration of 60mg/mL (I didn't went higher). Its predicted to have a lot of beta-strands (according to CD-spectra and secondary structure predictions). So here are my questions: - What is the molecular basis of phase separation? I mean what is going on at molecular level? I would suspect that my protein is not soluble in a PEG environment, is this correct? - What can I do to prevent my protein or buffer (?) going into phases? Is it temperature dependent? Are there additives I can add? Do I need to lower the salt concentration? - Are there examples (some of your personal experience) where phase separation was a good thing? For your record: the protein is in a 150mM NaCl, 20mM HEPES pH7.5 buffer and the pI is 5-6. It is cloned with a his-tag (but cleaving the his-tag didn't change much). Best Regards, Ruben Ruben Van der Meeren Ghent University L-ProBE, hoogbouw, verdiep 5 K. L. Ledeganckstraat 35 9000 Ghent (Belgium) E-Mail: ruben.vandermee...@ugent.be
[ccp4bb] creat a model with insertion
Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] creat a model with insertion
use coot: calculate - model/fit/refine - add terminal residue. this will add alanines. you can change these to glycines with the simple mutate option in the same pop up menu. best, HErman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Qing Chen Sent: Thursday, January 13, 2011 11:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] creat a model with insertion Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] creat a model with insertion
Let's say I need to insert two G between resi 98 and 99. I tried in the way as Herman suggest: break the chain in the domain boundary, add terminal residues at the N-terminus of the 2nd domain. But then there is no space to accommodate the inserted residues, the two glycerin backbones overlap with resi 97 and 98. Is there a way to shift the whole 1st domain a bit ? On Thu, Jan 13, 2011 at 12:16 PM, herman.schreu...@sanofi-aventis.comwrote: use coot: calculate - model/fit/refine - add terminal residue. this will add alanines. you can change these to glycines with the simple mutate option in the same pop up menu. best, HErman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Qing Chen *Sent:* Thursday, January 13, 2011 11:58 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] creat a model with insertion Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] creat a model with insertion
In that case, I would split your pdb file in two parts: Nterm to 101 and 99 to Cterm. With the rotate/translate zone option, you can move one complete domain to have residue 101 of the Nterm domain overlap with residue 99 of the Cterm domain. In a nex step you will have to delete residue 101 of the Nterm domain, which was only there as a help for the superposition, mutate residues 99 and 100 of the Nterm domain to glycine, renumber the Cterm to start at 101 and combine both pdb files again. Finally, you will have to regularize the link. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Qing Chen Sent: Thursday, January 13, 2011 12:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] creat a model with insertion Let's say I need to insert two G between resi 98 and 99. I tried in the way as Herman suggest: break the chain in the domain boundary, add terminal residues at the N-terminus of the 2nd domain. But then there is no space to accommodate the inserted residues, the two glycerin backbones overlap with resi 97 and 98. Is there a way to shift the whole 1st domain a bit ? On Thu, Jan 13, 2011 at 12:16 PM, herman.schreu...@sanofi-aventis.com wrote: use coot: calculate - model/fit/refine - add terminal residue. this will add alanines. you can change these to glycines with the simple mutate option in the same pop up menu. best, HErman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Qing Chen Sent: Thursday, January 13, 2011 11:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] creat a model with insertion Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] creat a model with insertion
Hello Qing Chen, if by 'shift' you mean the actual coordinates you can do in coot as Herman describes. If you want to shift the sequence numbering: Coot also has an option to renumber a sequence by an offset and the offset can also be negative. Cheers, Tim On Thu, Jan 13, 2011 at 12:27:49PM +0100, Qing Chen wrote: Let's say I need to insert two G between resi 98 and 99. I tried in the way as Herman suggest: break the chain in the domain boundary, add terminal residues at the N-terminus of the 2nd domain. But then there is no space to accommodate the inserted residues, the two glycerin backbones overlap with resi 97 and 98. Is there a way to shift the whole 1st domain a bit ? On Thu, Jan 13, 2011 at 12:16 PM, herman.schreu...@sanofi-aventis.comwrote: use coot: calculate - model/fit/refine - add terminal residue. this will add alanines. you can change these to glycines with the simple mutate option in the same pop up menu. best, HErman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Qing Chen *Sent:* Thursday, January 13, 2011 11:58 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] creat a model with insertion Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?
: Subject: Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein? My knowledge on this is probably quite out of date by now, but some years ago there was a lot of research on this topic because such surfaces are important in electrostatics and implicit solvation models (calculating surface area) as well as molecular graphics. I think the most widely-used definition of a solvent-accessible surface is Lee-Richards surface in which a solvent-sized sphere is rolled along the surface of the protein. Surface is therefore rigorously defined as a piecewise collection of convex and concave patches of spheres and tori. It was Connolly who implemented (and sold) a practical algorithm for computing these surfaces. They were even known as Connolly surfaces and rendered as dots before modern computing hardware allowed for rendering surfaces. Several groups have developed high-efficiency versions of the calculation. Harold Scheraga's group, for example, has some FORTRAN code for this. Fred Brook's virtual reality group also developed a high-effeciency parallel version (Varshney was the guy's name I think) in C. There have been many approximations over the years I think ... but you asked about analytical models. The these algorithms are non trivial. That's a understatement. And there is actually a mathematical ambiguity in the surface definition itself. The Varshney code is freely available ... I received email permission from both Varshney and his thesis advisor to freely distribute the code. I even offered it to Warren Delano years ago when he was writing Pymol, but he refused to include it because he felt there still might be legal issues that would effect Pymol. So ... Pymol contains only a somewhat improvised an non-rigorous surface algorithm (last time I looked). Fine for graphics of course. en.wikipedia.org/wiki/Accessible_surface_areahttp://en.wikipedia.org/wiki/Accessible_surface_area Richard On Jan 13, 2011, at 1:00 AM, Francois Berenger wrote: Hello, Does someone know some good articles on this particular topic? I'd like to implement the thing myself, however if there is a good software doing the job (with readable source code), I might use and cite it. Best regards, Francois.
[ccp4bb] Older cryocooling system
Would anybody offer an older crystal cooling system to go? -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
Re: [ccp4bb] creat a model with insertion
Hi Chen, Check SPDBViewer if it is of some help to you! Gauri On Thu, Jan 13, 2011 at 5:57 AM, Qing Chen qchen.c...@googlemail.comwrote: Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] Phase Separation
Hi Ruben, Timing is everything - We are just going through the proofs of a paper entitled What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments by Luft, Wolfley and Snell to appear shortly in Crystal Growth and Design. In putting this together we found a number of useful references related to the phase separation phenomena - temperature may be a very useful variable to try. To quote the relative paragraphs and hope we don't start a huge discussion (with references to the figures in the paper removed); There are protein-rich and protein-poor liquid phases. Protein concentrations of 400mg/mL have been measured in the protein-rich phase, a concentration comparable to that found in crystals. (1) Experimental and theoretical studies demonstrate the formation of immiscible liquid-liquid (L-L) phase separation in the metastable region of the phase diagram forms only where there are short range, and/or highly anisotropic interactions between protein molecules, with further experimental evidence that demonstrates this region is connected with conditions for growing crystals. (2) When the temperature of crystallization is near or below the formation temperature of a metastable, immiscible L-L phase separation, at high levels of supersaturation, experimental data and numerical simulations support a two-step, non-classical nucleation process.(2) In this mechanism a protein-rich liquid phase first forms. Nucleation takes place from this phase followed by initial growth of the nuclei sometimes into the protein-rich and other times into the protein-poor environment. Haas and Drenth(2) suggest that this growth mechanism can lead to fewer crystal defects and more rapid crystal growth as molecules in the concentrated liquid protein phase that surrounds the crystal are not driven to the surface of the crystal by diffusion and therefore misaligned molecules can be more readily exchanged. Literature also supports that it is not the higher protein concentration within the coacervate droplets or the molecular fluidity that may initiate nucleation but rather an interface effect between the dense liquid of high-protein concentration in the droplet and the immiscible surrounding liquid of low-protein concentration.(3) When a L-L phase separation is observed ... if one phase is protein-rich and the other protein-poor, then the system is very close to conditions that have the potential to produce crystals. If the protein contains tryptophan residues, then the presence of a protein-rich phase can be verified using UV fluorescence, Crystals will sometimes form from the dense liquid phase without intervention; As is the case with metastable conditions, this protein-rich immiscible liquid phase can be used for seeding.(4) The other useful and effective option to induce crystal formation is to drive the system towards a higher level of supersaturation, the labile state, using temperature. The rationale for this approach is to increase the attraction between protein molecules by decreasing the temperature.(5) However, this process will be dependent upon the solubility properties of the protein/solvent. Protein solubility is dictated by the combination of the protein and its chemical environment. The same protein can have increased solubility at higher temperatures in one chemical environment, and lower temperatures in a different chemical environment. If the protein/solvent is more soluble at higher temperatures and L-L phase separation is seen in the drop, then decreasing the temperature will drive the system towards a higher level of supersaturation. The opposite applies in cases where the protein/solvent exhibits retro-solubility, i.e. the protein is more soluble at lower temperatures. In this case the experiments would be moved to a higher temperature environment, or set up at a higher temperature in a replicate experiment. 1. Kuznetsov, Y. G.; Malkin, A. J.; McPherson, A. Journal of Crystal Growth 2001, 232, 30-39. 2. Haas, C.; Drenth, J. Journal of Physical Chemistry B 2000, 104, 368-377. 3. Vekilov, P. G. Crystal Growth Design 2004, 4, 671-685. 4. Bergfors, T. J Struct Biol 2003, 142, 66-76. 5. Dumetz, A. C.; Chockla, A. M.; Kaler, E. W.; Lenhoff, A. M. Biophys J 2008, 94, 570-583. Hope this helps, Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Senior Scientist, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ruben Van der Meeren Sent: Thursday, January 13, 2011 4:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Phase Separation Dear
Re: [ccp4bb] Phase Separation
It sounds a bit silly after that nice theoretical discussion, but I would try poking the existing oily blobs with a hair. Since you may be close to xtal conditions, stirring up the equilibrium a bit may help nucleate something. I've seen this work more than once, although usually with things where we'd seen some sign of crystals already. Also, try new drops with lower [PEG]. Good luck! = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Thu, 13 Jan 2011 12:11:54 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Edward Snell esn...@hwi.buffalo.edu) Subject: Re: [ccp4bb] Phase Separation To: CCP4BB@JISCMAIL.AC.UK Hi Ruben, Timing is everything - We are just going through the proofs of a paper entitled What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments by Luft, Wolfley and Snell to appear shortly in Crystal Growth and Design. In putting this together we found a number of useful references related to the phase separation phenomena - temperature may be a very useful variable to try. To quote the relative paragraphs and hope we don't start a huge discussion (with references to the figures in the paper removed); There are protein-rich and protein-poor liquid phases. Protein concentrations of 400mg/mL have been measured in the protein-rich phase, a concentration comparable to that found in crystals. (1) Experimental and theoretical studies demonstrate the formation of immiscible liquid-liquid (L-L) phase separation in the metastable region of the phase diagram forms only where there are short range, and/or highly anisotropic interactions between protein molecules, with further experimental evidence that demonstrates this region is connected with conditions for growing crystals. (2) When the temperature of crystallization is near or below the formation temperature of a metastable, immiscible L-L phase separation, at high levels of supersaturation, experimental data and numerical simulations support a two-step, non-classical nucleation process.(2) In this mechanism a protein-rich liquid phase first forms. Nucleation takes place from this phase followed by initial growth of the nuclei sometimes into the protein-rich and other times into the protein-poor environment. Haas and Drenth(2) suggest that this growth mechanism can lead to fewer crystal defects and more rapid crystal growth as molecules in the concentrated liquid protein phase that surrounds the crystal are not driven to the surface of the crystal by diffusion and therefore misaligned molecules can be more readily exchanged. Literature also supports that it is not the higher protein concentration within the coacervate droplets or the molecular fluidity that may initiate nucleation but rather an interface effect between the dense liquid of high-protein concentration in the droplet and the immiscible surrounding liquid of low-protein concentration.(3) When a L-L phase separation is observed ... if one phase is protein-rich and the other protein-poor, then the system is very close to conditions that have the potential to produce crystals. If the protein contains tryptophan residues, then the presence of a protein-rich phase can be verified using UV fluorescence, Crystals will sometimes form from the dense liquid phase without intervention; As is the case with metastable conditions, this protein-rich immiscible liquid phase can be used for seeding.(4) The other useful and effective option to induce crystal formation is to drive the system towards a higher level of supersaturation, the labile state, using temperature. The rationale for this approach is to increase the attraction between protein molecules by decreasing the temperature.(5) However, this process will be dependent upon the solubility properties of the protein/solvent. Protein solubility is dictated by the combination of the protein and its chemical environment. The same protein can have increased solubility at higher temperatures in one chemical environment, and lower temperatures in a different chemical environment. If the protein/solvent is more soluble at higher temperatures and L-L phase separation is seen in the drop, then decreasing the temperature will drive the system towards a higher level of supersaturation. The opposite applies in cases where the protein/solvent exhibits retro-solubility, i.e. the protein is more soluble at lower temperatures. In this case the experiments would be moved to a higher temperature environment, or set up at a higher temperature in a replicate experiment. 1. Kuznetsov, Y. G.; Malkin, A. J.; McPherson, A. Journal of Crystal
[ccp4bb] Corrections in Sherwood Cooper
One of my colleagues asked if I could post the following to the ccp4bb: Thanks to very helpful feedback, there is now a fairly comprehensive set of curations for the new 'Crystals, X-rays and Proteins' (Sherwood and Cooper) at the following link: http://www.ucl.ac.uk/~rmhajc0/ The first 6 are the most important!
[ccp4bb] Postdoctoral Positions in Ion Channel Structure at UCSF
POSTDOCTORAL POSITIONS, MINOR LAB UNIVERSITY OF CALIFORNIA SAN FRANCISCO (UCSF) Structure, Function, and Regulation of Ion Channels Postdoctoral positions in Ion Channel Structural Biology are available immediately for highly motivated individuals with a strong interest in integrated approaches to problems in ion channel structure, function, and regulation in the lab of Prof. Dan Minor at the University of California, San Francisco (UCSF). The Minor Lab merges structural, biochemical, genetic, and electrophysiological methods to dissect the mechanism of complex protein machines involved in electrical signaling. More information is available at the lab website: http://www.cvri.ucsf.edu/~dminor/ The positions offer ideal opportunities for experienced crystallographers interested in continuing structural studies, but who would like to complement his or her expertise with other, diverse tools for understanding membrane protein function and mechanism. Available projects also include investigation of full-length membrane protein structures. The fellows will benefit from both the outstanding environment in the lab and the highly collaborative UCSF community. The lab has extensive crystallographic resources, including (as part of the UCSF crystallography group) two R-axis IV systems and regular access to synchrotron beamline 8.3.1 at the nearby Advanced Light Source (ALS) in Berkeley. Candidates should have (or expect) an Ph.D. or M.D. and should have experience in protein purification, crystallization, and structure determination. Interested individuals should send a current CV to Prof. Daniel Minor at daniel.mi...@ucsf.edu Dan Minor, Ph.D. Associate Professor Cardiovascular Research Institute Departments of Biochemistry Biophysics, and Cellular Molecular Pharmacology California Institute for Quantitative Biomedical Research University of California, San Francisco Mailing/FEDEX/UPS address: Cardiovascular Research Institute Box 3122 University of California San Francisco 555 Misson Bay Blvd. South. Rm 452Z San Francisco, CA 94158-9001 Email: daniel.mi...@ucsf.edu Phone: 415-514-2551 Fax: 415-514-2550 Web: http://www.cvri.ucsf.edu/~dminor
[ccp4bb] Postdoctoral Positions in Ion Channel Structure at UCSF- CVRI - available for 2011
POSTDOCTORAL POSITIONS, MINOR LAB - Available for start in 2011 CARDIOVASCULAR RESEARCH INSTITUTE UNIVERSITY OF CALIFORNIA SAN FRANCISCO (UCSF) Structure, Function, and Regulation of Ion Channels Postdoctoral positions in Ion Channel Structural Biology are available immediately for highly motivated individuals with a strong interest in integrated approaches to problems in ion channel structure, function, and regulation in the lab of Prof. Dan Minor at the University of California, San Francisco (UCSF). The Minor Lab merges structural, biochemical, genetic, and electrophysiological methods to dissect the mechanism of complex protein machines involved in electrical signaling. More information is available at the lab website: http://www.cvri.ucsf.edu/~dminor/ The positions offer ideal opportunities for experienced crystallographers interested in continuing structural studies, but who would like to complement expertise with other, diverse tools for understanding membrane protein function and mechanism. Available projects also include investigation of full-length membrane protein structures. The fellows will benefit from both the outstanding environment in the lab and the highly collaborative UCSF community. The lab has extensive crystallographic resources, including (as part of the UCSF crystallography group) two R-axis IV systems and regular access to synchrotron beamline 8.3.1 at the nearby Advanced Light Source (ALS) in Berkeley. The lab has recently relocated to the new Smith Cardiovascular Research Building on the UCSF Mission Bay Campus http://www.hawkeyeaerial.com/UCSF_083110/ Candidates should have (or expect) an Ph.D. or M.D. and should have experience in protein purification, crystallization, and structure determination. Interested individuals should send a current CV to Prof. Daniel Minor at daniel.mi...@ucsf.edu Dan Minor, Ph.D. Associate Professor Cardiovascular Research Institute Departments of Biochemistry Biophysics, and Cellular Molecular Pharmacology California Institute for Quantitative Biomedical Research University of California, San Francisco Mailing/FEDEX/UPS address: Cardiovascular Research Institute Box 3122 University of California San Francisco 555 Misson Bay Blvd. South. Rm 452Z San Francisco, CA 94158-9001 Email: daniel.mi...@ucsf.edu Phone: 415-514-2551 Fax: 415-514-2550 Web: http://www.cvri.ucsf.edu/~dminor
[ccp4bb] Looking for the following values...
Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? Thanks in advance, -Jon
Re: [ccp4bb] Looking for the following values...
Hi, Generally if we use CCP4i we can find these details easily in scala log files. Gauri On Thu, Jan 13, 2011 at 2:48 PM, J. Fleming jonathan...@hotmail.com wrote: Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? Thanks in advance, -Jon
Re: [ccp4bb] Looking for the following values...
On 1/13/11 2:48 PM, J. Fleming wrote: Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) There is no criterion for sigma F applied in Denzo/HKL2000. Not least of all because data processing programs like Denzo and Scalepack work with intensities and not structure factor moduli. The default Sigma(I) cutoff is -3 See: http://www.hkl-xray.com/hkl_web1/hkl/Scalepack_Keywords.html (keyword SIGMA CUTOFF) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) If your refinement program does not write it into the header of the PDB file, and the description of the value does not make immediate sense to you, omit it. Some of the requested values are defined rather vaguely. A field matching this name doesn't show up in the REMARK 3 refinement template for PHENIX-derived PDB files. (http://www.wwpdb.org/docs.html) I haven't deposited lately but if I were to hazard a *guess* it might approximate to the number of reflections you would have used in refinement if you hadn't applied magnitude or sigma(F) cutoffs and prior to PHENIX rejecting reflections as gross statistical outliers. One straightforward way to get this number would be to use CAD to write a new MTZ file containing only reflections within the resolution limits used in refinement, and look in the log file to see what the output reflection count was. Assuming, of course, that the cell dimensions defined in your MTZ file are the same ones that you used in refinement. Refinement programs vary in their policy about handling reflections with |F|=0. The loss of reflections would manifest in a difference between the completeness in data collection and the completeness in refinement. Phil Jeffrey Princeton
Re: [ccp4bb] Looking for the following values...
Hi Jon, a partial answer: 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? a PDB file header with phenix.refine refined structure contains this number under REMARK 3 records. Can't ADIT extract this information automatically? Look in your PDB file for something like that: REMARK 3 NUMBER OF REFLECTIONS : 15114 Although, it's derivable information, meaning that if you deposit the data used for refinement then you should be safe. Pavel.
Re: [ccp4bb] Looking for the following values...
Hi Jon - - Observed criterion sigma(F) and sigma(I) means what cutoff did your data processing package use to consider a reflection not observed, i.e. throw it out? If you processed your data with Scalepack/HKL2000, the default cutoff is on I, and it's negative 3 sigma. There is no cutoff on F. So you would enter -3 for the sigma(I) entry and leave the sigma(F) entry blank. I don't think that both of these entries are required. - The difference between all reflections and observed reflections is, as far as I can tell, that all reflections indicates the number of mathematically possible unique reflections given your space group, unit cell, and diffraction limits. I generally just leave these entries blank where I can, and where it's required, I divide my number of observed reflections by my percent completeness - for example, a dataset with 31984 unique observed reflections (you can get these numbers from the scaling log files of Scalepack or scala) with 99.8% completeness would give you (31984/0.998)=32048 as the number of all reflections. Hope that helps, Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165 _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of J. Fleming Sent: Thursday, January 13, 2011 2:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Looking for the following values... Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? Thanks in advance, -Jon
Re: [ccp4bb] Looking for the following values...
I find this confusing also! Phil Jeffrey wrote: On 1/13/11 2:48 PM, J. Fleming wrote: Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) There is no criterion for sigma F applied in Denzo/HKL2000. Not least of all because data processing programs like Denzo and Scalepack work with intensities and not structure factor moduli. Both are marked as required, but if you leave one blank the deposition will go through anyway and the annotator will not have a problem with this. Just as synthetic source and engineered source are required, but you leave them blank or NULL if your protein is from a natural source. The default Sigma(I) cutoff is -3 See: http://www.hkl-xray.com/hkl_web1/hkl/Scalepack_Keywords.html (keyword SIGMA CUTOFF) This is more complicated if there are partials - The -3 Sigma cut-off applies to measurements measurements can be full or partial (I think). I believe the reflection is rejected if any of its partials are rejected- (although theoretically it should be possible to approximate an intensity from intensity of one of its partials) The number rejected will be greater if only one of the partials needs to fail the sigma cutoff than if they are all added up before the cutoff is applied 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I think in refinement this just means test + working reflections? In data reduction it is more complicated. I think you have the following: measurements - number of spots for which denzo recorded a complete reflection or a full set of partials (different from measurements above?) reflections, possible - Theoretical number unique reflections in range reflections, measured - Unique reflections with at least one measurement reflections, observed - whats left, after aplying rejection criterion during data reduction. I would say once a reflection has been measured, it has already passed the -3sigma cutoff, we never reject a measured reflection. reflections used in refinement - after applying resolution and sigma cutoffs in refinement (Don't take my word for this- I'm confused, as you can see. Hoping for clarification) Another question- it is often suggested to use I/sigI = 2 in a shell as the criterion for resolution cutoff. And I/sigI is one of the parameters needed for deposition. But is this the average I/sigI for individual measurements, which (I think) is what you can estimate from the last table of scalepack output, or the average of I/SigI for the reduced unique reflections, which you could get from a table in truncate log file? Sigma I for the reduced reflection should be smaller than sigmaI for the individual measurements, as standard error of the mean is smaller than standard deviation of the measurement.
