Re: [ccp4bb] validating ligand density
Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan
Re: [ccp4bb] validating ligand density
Dear Srinivasan, The first thing I would do is to look very carefully at the electron density maps: Does it look like a bunch of water molecules, or is continuous density present? Could it be some buffer component (Tris, Hepes, sulfate, DMSO etc) or the counter ion of your ligand or the cryoprotectant (e.g. glycerol)? Does the protein have disordered residues e.g. at the N- or C-terminus, which could bind to the binding site? Same is true for sugars if your protein is glycosylated. If you are crystallizing a protease, autolysis during crystallization may have released short peptides which may bind to the ligand binding site. Is the binding mode of your ligand disordered? Would the electron density be better explained if you fit the ligand in two alternative conformations? If your ligand is bound at low occupancy, something else (e.g. waters, sulfate) may be bound in those binding sites where the ligand is not present. Also side chains may adopt a different conformation in the absence of the ligand. In this case one should model all alternatives. After this analysis, one usually ends up with a limited number of alternatives (e.g. ligand is bound, bunch of water molecules, something else is bound). Most informative is to fit and refine all (in this case three) possibilities and look which electron density map looks most convincing. I would also calculate the real space correlation coefficient for each alternative. If possible, I would also compare the electron density with the electron density of apo-crystals (not cocrystallized or soaked with the ligand). If the same density is present there, it is not your ligand but something else. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 11:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan
Re: [ccp4bb] validating ligand density
I guess I have known lots of incorrectedly fitted ligands, and am never sure quite how to be confident they are correct. Your resolution is good, so one hopes the density is pretty clear? Common sources of error. By far the most common is caused by having an incorrect dictionary. Look very carefully at what is expected - is the chirality correct; the planarity? the bonding - does it fit what the chemists expect?? Second - over interpreting the density - some ligands have extremely wobbly bits, and I am never sure what to do about them - I tend to get overoptimistic, but with your data the B factors should help indicate such sections. I set the occupancies to 0.00 for those bits - re-refine and look at the difference maps again for verification - coot is very good to fitting ligands if there is density to fit to. Make sure the B factor restraints within the ligand are not too tight . Sometimes if your ligand contains S or P and the data is good enough you can use the anomalous difference fourier maps to see if there are peaks over the expected scatterers. (This check is a bit of a pain to do - you have to use CAD to add DANO SIGDANO to the REFMAC output, then do the map and peak search) Eleanor On 12 March 2013 10:02, herman.schreu...@sanofi.com wrote: ** Dear Srinivasan, The first thing I would do is to look very carefully at the electron density maps: Does it look like a bunch of water molecules, or is continuous density present? Could it be some buffer component (Tris, Hepes, sulfate, DMSO etc) or the counter ion of your ligand or the cryoprotectant (e.g. glycerol)? Does the protein have disordered residues e.g. at the N- or C-terminus, which could bind to the binding site? Same is true for sugars if your protein is glycosylated. If you are crystallizing a protease, autolysis during crystallization may have released short peptides which may bind to the ligand binding site. Is the binding mode of your ligand disordered? Would the electron density be better explained if you fit the ligand in two alternative conformations? If your ligand is bound at low occupancy, something else (e.g. waters, sulfate) may be bound in those binding sites where the ligand is not present. Also side chains may adopt a different conformation in the absence of the ligand. In this case one should model all alternatives. After this analysis, one usually ends up with a limited number of alternatives (e.g. ligand is bound, bunch of water molecules, something else is bound). Most informative is to fit and refine all (in this case three) possibilities and look which electron density map looks most convincing. I would also calculate the real space correlation coefficient for each alternative. If possible, I would also compare the electron density with the electron density of apo-crystals (not cocrystallized or soaked with the ligand). If the same density is present there, it is not your ligand but something else. Good luck! Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of * R.Srinivasan *Sent:* Monday, March 11, 2013 11:03 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan
Re: [ccp4bb] validating ligand density
One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan
Re: [ccp4bb] validating ligand density
One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] validating ligand density
Dear Jacob, You are overinterpreting, the statement is about judging, not proving a hypothesis. I am sure Mr. Edwards judged his statement to be ok. ;-) Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Tuesday, March 12, 2013 3:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] validating ligand density
Dear Jacob, You are overinterpreting, the statement is about judging, not proving a hypothesis. I am sure Mr. Edwards judged his statement to be ok. I guess there is a good likelihood that you are right, but who am I to judge? JPK Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Jacob Keller *Sent:* Tuesday, March 12, 2013 3:44 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] validating ligand density One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org *** -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
[ccp4bb] FW: [ccp4bb] validating ligand density
You are the one who should judge your statement, but it looks plausible to me. Now that I think of it: why do we need referees if every scientist should judge their own hypothesis? Publication will be a lot faster if we no longer need to heed the remarks of some grumpy referees and send in revision after revision. Also the number of publications will increase significantly if every scientist is allowed to judge their own papers! HS From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Tuesday, March 12, 2013 4:14 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] validating ligand density Dear Jacob, You are overinterpreting, the statement is about judging, not proving a hypothesis. I am sure Mr. Edwards judged his statement to be ok. I guess there is a good likelihood that you are right, but who am I to judge? JPK Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Tuesday, March 12, 2013 3:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure
Re: [ccp4bb] [ccp4bb] validating ligand density
Going back to the initial question. I would recommend looking at AFITT http://www.eyesopen.com/afitt Works like a dream (in certain cases). Jürgen P.S. I wish I had some stocks from them but I don't .. 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://lupo.jhsph.edu On Mar 12, 2013, at 11:25 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: You are the one who should judge your statement, but it looks plausible to me. Now that I think of it: why do we need referees if every scientist should judge their own hypothesis? Publication will be a lot faster if we no longer need to heed the remarks of some grumpy referees and send in revision after revision. Also the number of publications will increase significantly if every scientist is allowed to judge their own papers! HS From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Tuesday, March 12, 2013 4:14 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] validating ligand density Dear Jacob, You are overinterpreting, the statement is about judging, not proving a hypothesis. I am sure Mr. Edwards judged his statement to be ok. I guess there is a good likelihood that you are right, but who am I to judge? JPK Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Tuesday, March 12, 2013 3:44 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero compounds in the PDB were so incredibly 'not there' that Refmac would crash (that bug seems to be fixed in the latest version). HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.Srinivasan Sent: Monday, March 11, 2013 23:03 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating ligand density Hello all, We co-crystallized an inactive variant of our enzyme in the presence of substrate and have determined the structure at 1.85A. Now, we want to validate the fitting of the ligand into the electron density. We tried validating using the difference map (2Fo-Fc) after refining the structure without the ligand. But, it is still a bit inconclusive if the density fits the ligand. It would be very kind to know if there are tools for validating this electron density. We were excited about twilight but turns out it can only be used with deposited structure. We will appreciate your help and suggestions. Many thanks, Srinivasan -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org *** -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email:
Re: [ccp4bb] [ccp4b] statistical or systematic? bias or noise?
Hi Ed, You can have both types of error in a single experiment, however you cannot determine statistical (precision or as Ian says uncontrollable) error with one experiment. The manufacturer will usually give some specs on the pipette, 6ul +/- 1ul. In order to verify the specs you would need to perform many pipetting experiments. But even if the manufacturer does not give any specs you still know that the pipette is not perfect and there will be a statistical error, you just do not know what it is. In theory, accuracy or bias could be determined with one experiment. Lets say you thought you had a 6ul pipette but actually it was a 12ul pipette. If you then compare the 'new' pipette against a standard you could tell if it was inaccurate. Of course normally you would repeat this experiment as well because of statistical error. If detected bias can be removed. Systematic error may not be so easily detected. What if the standard is also biased. Adam One can say it's inaccuracy when it is not estimated and imprecision when it is. Or one can accept Ian's suggestion and notice that there is no fundamental difference between things you can control and things you can potentially control.
Re: [ccp4bb] statistical or systematic? bias or noise?
On Mon, 11 Mar 2013 11:46:03 -0400, Ed Pozharski epozh...@umaryland.edu wrote: ... Notice that I only prepared one sample, so if on that particular instance I picked up 4.8ul and not 5.0ul, this will translate into systematically underestimating protein concentration, even though it could have equally likely been 5.2ul. Within the framework of such a short Materials and Methods description, the latter is _not_ a systematic error; rather, you are sampling (once!) a statistical error component. If possible, you should repeat the experiment and find out the magnitude of your pipetting error. If impossible, you can only estimate it (making reasonable assumptions based on past experiments). Of course, if - in repeated experiments - your pipetting more often gives (e.g.) lower volumes than 5.0 ul, then some kind of systematic error must be the reason. Systematic error in most cases has the property that it (on average) changes the result towards one side, whereas statistical error should not change the mean value. It is one of the goals of an experiment to identify all sources of systematic error, and to either model or eliminate them. If you are able to identify and model the systematic error, then you can convert noise to signal. best, Kay
[ccp4bb] Research Support Specialist - Yale University
Dear All, For any small molecule crystallographers who may read this board, the Yale Chemistry Biophysical Instrumentation Center has a position available. Besides performing service crystallography the successful applicant will cooperate with other CBIC staff in the maintenance of analytical equipment and some other duties. To view the opening visit Yale's external Careers site: http://www.yale.edu/hronline/careers/application/external/index.html Search for STARS Requisition number 20613BR Best wishes, Richard Baxter -- Asst. Prof. in Chemistry = Yale University PO Box 208107 New Haven, CT 06520-8107 Tel: +1 203 432 9576 Fax: +1 203 432 6144 www.baxterlab.org =
Re: [ccp4bb] validating ligand density
Thank you very much to all those who suggested a way out for our situation. We have so far refined the occupancies on phenix and the ligand shows an uniform occupancy of 0.8 post the refinement. The B-factors of the ligand are around 20 (atleast for the regions with a well defined electron density), which is slightly lower than the average B-factors of the whole structure which is 24. We have a few poorly defined regions in our electron density. This was the starting point of our problem and it remains to be a problem. @ Robbie --- we would like to run EDSTAT on CCP4 but we dont find the program in both 6.3.0 and 6.3.1 versions. It would be kind to know if we are doing something wrong to not find it on the ensuite. @ Herman We did add the cryo from the crystallisation condition as another strategy but that also doesnt look too convincing. There is just that enough more to the density to think its our substrate. The density also does not compare well with the apo structures. @ Eleanor We set the occupancies to zero and refined the structure but we did not get any conclusive answers from it. We have a continious density for the best part of the ligand; but as you mentioned a few carbon atoms which are wobbly are poorly defined. We will look carefully into the geometrical restraintsas you suggested. Thank you all again forthe suggestions! Srinivasan From: Bosch, Juergen jubo...@jhsph.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, 12 March 2013 4:32 PM Subject: Re: [ccp4bb] [ccp4bb] validating ligand density Going back to the initial question. I would recommend looking at AFITT http://www.eyesopen.com/afitt Works like a dream (in certain cases). Jürgen P.S. I wish I had some stocks from them but I don't .. 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://lupo.jhsph.edu On Mar 12, 2013, at 11:25 AM, herman.schreu...@sanofi.com herman.schreu...@sanofi.com wrote: You are the one who should judge your statement, but it looks plausible to me. Now that I think of it: why do we need referees if every scientist should judge their own hypothesis? Publication will be a lot faster if we no longer need to heed the remarks of some grumpy referees and send in revision after revision. Also the number of publications will increase significantly if every scientist is allowed to judge their own papers! HS From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Tuesday, March 12, 2013 4:14 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] validating ligand density Dear Jacob, You are overinterpreting, the statement is about judging, not proving a hypothesis. I am sure Mr. Edwards judged his statement to be ok. I guess there is a good likelihood that you are right, but who am I to judge? JPK Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Tuesday, March 12, 2013 3:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density One final quote that is not in the twilight paper summarizes it nicely: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. There must be a lot of thinking behind this statement--while it seems plausible, it seems far from proven prima facie. Also, it assumes that the scientist is not a statistician. Jacob Btw, the book is good reading. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] validating ligand density Dear Srinivasan, Although the Twilight program can only look at deposited PDB entries, the tips about ligand validation in the paper are very useful. I suggest you start from there. You can use EDSTATS in CCP4 to get real-space validation scores. Also look at the difference map metrics it gives (and the maps themselves of course), they will tell you whether you misidentified your ligand. Occupancy refinement in Refmac can also help you: if the occupancy drops a lot something is wrong. That can be partial binding (not that much of a problem) or worse, a ligand that isn't there. By the way, I've been playing with that recently and some ligands/hetero
[ccp4bb] Frontiers in Neutron Structural Biology Workshop - Important Dates
Frontiers in Neutron Structural Biology Oak Ridge National Laboratory Spallation Neutron Source April 16-18, 2013 REMINDER - Registration deadline soon approaching. Deadline Dates: The deadline for application is March 15, 2013, unless you qualify for one of the following exceptions: The application deadline can be extended to April 1, 2013 if: * You are making your own travel arrangements and not requesting travel funds from workshop sponsors, and * You have a current ORNL badge, and * You are a U.S. citizen. Application: Everyone attending the workshop must register. PLEASE NOTE: Day 3 will focus on panel discussion and preparation of the workshop report. Panelists will include invited speakers and facility experts, but all participants are welcome to attend. More information can be found at this link https://neutrons.ornl.gov/conf/frontier2013. Frontiers in Neutron Structural Biology Oak Ridge National Laboratory Spallation Neutron Source April 16-18, 2013 This meeting will bring together scientists to discuss new opportunities for biomedical research at the two advanced neutron user facilities (SNS and HFIR) at the Department of Energy's (DOE) Oak Ridge National Laboratory (ORNL). Users now have access to some of the world's most intense neutron beamlines for studying the structure, function, and dynamics of complex biological systems. ORNL plans to establish and operate a biomedical neutron technology research center (BNTRC) that will integrate and develop neutron scattering technologies with high performance supercomputing and biomolecular synthesis and deuterium-labeling. Participants will identify new scientific challenges and lines of biomedical inquiry that will drive the development and integration of these leading-edge technologies. An important function of the BNTRC will be to provide training, access, and expert assistance in these technologies to biomedical researchers. In a satellite meeting, the developers of the comprehensive software suite Phenix (http://www.phenix-online.org/)http://www.phenix-online.org/ for macromolecular structure determination will give a one-day workshop on the use of the software for macromolecular neutron crystallography (MNC). Participants are encouraged to bring a laptop for the afternoon tutorial session. MNC is in a period of expansion at the moment; in North America the number of beamlines suitable for MNC is expected to quadruple over the next year. This workshop will introduce current and future MNC users to the new experimental beamlines at ORNL and provide a tutorial for structure refinement using PHENIX and nCNS. New developments will be described that greatly enhance structure refinement. Participants will be given real X-ray and neutron (XN) data and will be taken through joint XN structure refinement of proteins. Scholarships for attendance are available from: * Joint Institute for Neutron Scienceshttp://jins.tennessee.edu/ for those attendees from EPSCOR states (Alabama, Alaska, Arkansas, Delaware, Hawaii, Idaho, Iowa, Kansas, Kentucky, Louisiana, Maine, Mississippi, Montana, Nebraska, Nevada, New Mexico, North Dakota, Oklahoma, Rhode Island, South Carolina, South Dakota, Tennessee, Utah, Vermont, West Virginia, Wyoming, the Commonwealth of Puerto Rico, and the Virgin Islands). For details, please visit http://jins.tennessee.edu/epscor/index.html for the application form and contact Hope Moore at hmoo...@utk.edumailto:hmoo...@utk.edu. EPSCoR fellowship is federally funded through DOE and is unable to provide travel support for individuals employed by DOE institutions. * The ORAU Travel Grants Programhttp://www.orau.org/university-partnerships/faculty-student-programs/default.aspx provides up to $800 to facilitate travel by a faculty member from an ORAU Sponsoring or Associate Institution or Branch Campus. Visits can be to collaborate with researchers at ORNL, Y-12, ORAU laboratories or work sites, or another ORAU institution. To apply, visit the ORAU Members Universities pagehttp://www.orau.org/university-partnerships/members.aspx, find your school, and contact your member councilor, who can submit a proposal for travel funding to the ORAU University Partnerships Office through the Members Onlyhttp://www.orau.org/university-partnerships/members-only.aspx section of that site. More information can be found at this link https://neutrons.ornl.gov/conf/frontier2013.
