Re: [ccp4bb] validating ligand density
Hi Srinivasan, I always use edstats from the command line: 'edstats.pl HKLIN 101m_0cyc.mtz XYZIN 101m_0cyc.pdb'. I hadn't noticed it was well-hidden (or the limit case: not present) in CCP4i. I guess a feature request is in order. Cheers, Robbie Date: Wed, 13 Mar 2013 03:42:45 +0800 From: sreera...@yahoo.co.in Subject: Re: [ccp4bb] validating ligand density To: CCP4BB@JISCMAIL.AC.UK 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 restraints as you suggested. Thank you all again for the suggestions!Srinivasan From: "Bosch, Juergen" 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 AFITThttp://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, 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 R&D/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
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" 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, 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 R&D/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--
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, mailto:herman.schreu...@sanofi.com>> mailto: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 R&D/DE Cc: CCP4BB@jiscmail.ac.uk<mailto: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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Jacob Keller Sent: Tuesday, March 12, 2013 3:44 PM To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Robbie Joosten Sent: Tuesday, March 12, 2013 10:03 AM To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of > R.Srinivasan > Sent: Monday, March 11, 2013 23:03 > To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto: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 R&D/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
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 ***
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
> > 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
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
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, 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
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
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
[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
