[ccp4bb] AW: [ccp4bb] [ccp4bb] AW: [ccp4bb] uncertainites associated with intensities from twinned crystals; Sorry for HTML.
Dear Fulvio and others,
I do not understand this whole discussion. In case of perfectly twinned
crystals, it is impossible to derive a detwinned F1 and F2 from two
independent, but otherwise identical measurements. In this case, the only
signal is noise, and one could as well use a random generator to get the
detwinned data. It makes perfectly sense to me that in this case the
theoretical error would be infinite. In practical terms, since in case of
twinning intensities and not structure factors are added, the error cannot be
larger than twice the largest of the two measurements plus twice the error for
that measurement. There might be a formula to properly calculate this error.
My 2 cents,
Herman
-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jens
Kaiser
Gesendet: Donnerstag, 7. November 2013 08:29
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] AW: [ccp4bb] uncertainites associated with
intensities from twinned crystals; Sorry for HTML.
Tassos,
I'm no expert either, and there are caveats for using this formula on
correlated magnitudes. But I would assume that the intensities of twin related
reflections should be independent from each other (that's my understanding of
the sigmoid cumulative intensity distribution of twins). Thus, I think the
simple Gaussian error propagation should be applicable to uncertainty estimates
in detwinned intensities.
Cheers,
Jens
On Thu, 2013-11-07 at 08:09 +0100, Anastassis Perrakis wrote:
Dear Jens,
That formula for error propagation is correct for independent
measurements.
Does this assumption stand true for Intensities in twinning? I am no
expert, but I would think not.
Tassos
On 7 Nov 2013, at 7:53, Jens Kaiser wrote:
Fulvio, Tim,
error propagation is correct, but wrongly applied in Tim's
example.
s_f= \sqrt{ \left(\frac{\partial f}{\partial {x} }\right)^2 s_x^2 +
\left(\frac{\partial f}{\partial {y} }\right)^2 s_y^2 +
\left(\frac{\partial f}{\partial {z} }\right)^2 s_z^2 + ...} (see
http://en.wikipedia.org/wiki/Propagation_of_uncertainty#Simplificati
on) The uncertainty in a derived magnitude is always larger than any
individual uncertainty, so no subtraction, anytime. Furthermore, in
Tim's example you could end up with negative sigmas..
HTH,
Jens
On Thu, 2013-11-07 at 04:44 +0100, Tim Gruene wrote:
Dear Fulvio,
with simple error propagation, the error would be
sigma(I(h1)) = (1-α)sigma(Iobs(h1))-α*sigma(Iobs(h2))/(1-2α)
would it not?
Although especially for theoretical aspects you should be
concerned about division by zero.
Best,
Tim
On 11/06/2013 05:54 PM, Fulvio Saccoccia wrote:
Thank you for reply. My question mostly concern a theoretical
aspect rather than practical one. To be not misunderstood, what
is the mathematical model that one should apply to be able to
deal with twinned intensities with their errors? I mean,
I+_what? I ask this In order to state some general consideration
on the accuracy about the recovery the true intensities on
varying of alpha. Thanks Fulvio
Fulvio Saccoccia PhD Dept. of Biochemical Sciences Sapienza
University of Rome 5, Piazzale A. Moro 00185 phone +39
0649910556
Messaggio Originale Da: herman.schreu...@sanofi.com
Inviato: 06/11/2013, 17:25 A: CCP4BB@JISCMAIL.AC.UK Oggetto:
[ccp4bb] AW: [ccp4bb] uncertainites associated with intensities
from twinned crystals
Dear Fulvio, you cannot detwin perfectly twinned data with this
formula. The term (1-2α) becomes zero, so you are dividing by zero.
With good refinement programs (ShelX, Refmac), refinement is
done against twinned data, which is better than to detwin the
data with the formula you mention.
As I understand it, to get map coefficients, the calculated
contribution of the twin domain (Fcalc’s) is substracted from
Fobs (with the appropriate weighting factors), so what you see
in coot is detwinned electron density. In practical terms, the
only thing you have to do is to specify the TWIN keyword in Refmac.
Best regards, Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im
Auftrag von Fulvio Saccoccia Gesendet: Mittwoch, 6. November 2013 16:58
An:
CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] uncertainites associated
with intensities from twinned crystals
Dear ccp4 users
a question about the recovering of true intensities from
merohedral twinned crystal. Providing alpha and the twin
operator one should be able to recover the intensities from the
formulas:
I(h1) = (1-α)Iobs(h1)-αIobs(h2)/(1-2α)
I(h2) = -αIobs(h1)+(1+α)Iobs(h2)/(1-2α)
as stated in many papers and books*.
However I was wondering about the
Re: [ccp4bb] [ccp4bb] AW: [ccp4bb] uncertainites associated with intensities from twinned crystals; Sorry for HTML.
Dear all,
thank you for your reply. I would summarize my concerns and opinions,
so
far:
1) for QTLS (non-merohedral twinning - non intersecting lattices) I think one
should consider the variables as independent and random and it is possible to
recover the true intensities of a unique lattice from the stronger diffracting
one (see for example Jenni Ban, 2009, Acta D65, 101-111). Hence, the
quadratic formula (reported fomr Jens Kaiser) can be applied;
2) for TLS (merohedral twinning - perfectly overlapping spots) I think one
should not consider the two variable independent since they are related by
alpha (see the formulas I reported in my first message). In this case, I think
the right formula should be that reported by Tim Grune, that as far as I know
overestimates the true error but in this case the quadratic is not applicable.
Therefore, one would be prone to conclude that the uncertainties associated to
merohedral-twinned crystals are higher than regular crystals or non-merohedral
crystals. What's your opinion about?
In data mercoledì 6 novembre 2013 23:29:01, Jens Kaiser ha scritto:
Tassos,
I'm no expert either, and there are caveats for using this formula on
correlated magnitudes. But I would assume that the intensities of twin
related reflections should be independent from each other (that's my
understanding of the sigmoid cumulative intensity distribution of
twins). Thus, I think the simple Gaussian error propagation should be
applicable to uncertainty estimates in detwinned intensities.
Cheers,
Jens
On Thu, 2013-11-07 at 08:09 +0100, Anastassis Perrakis wrote:
Dear Jens,
That formula for error propagation is correct for independent
measurements.
Does this assumption stand true for Intensities in twinning? I am no
expert, but I would think not.
Tassos
On 7 Nov 2013, at 7:53, Jens Kaiser wrote:
Fulvio, Tim,
error propagation is correct, but wrongly applied in Tim's
example.
s_f= \sqrt{ \left(\frac{\partial f}{\partial {x} }\right)^2 s_x^2 +
\left(\frac{\partial f}{\partial {y} }\right)^2 s_y^2 +
\left(\frac{\partial f}{\partial {z} }\right)^2 s_z^2 + ...} (see
http://en.wikipedia.org/wiki/Propagation_of_uncertainty#Simplification)
The uncertainty in a derived magnitude is always larger than any
individual uncertainty, so no subtraction, anytime. Furthermore, in
Tim's example you could end up with negative sigmas..
HTH,
Jens
On Thu, 2013-11-07 at 04:44 +0100, Tim Gruene wrote:
Dear Fulvio,
with simple error propagation, the error would be
sigma(I(h1)) = (1-α)sigma(Iobs(h1))-α*sigma(Iobs(h2))/(1-2α)
would it not?
Although especially for theoretical aspects you should be concerned
about division by zero.
Best,
Tim
On 11/06/2013 05:54 PM, Fulvio Saccoccia wrote:
Thank you for reply. My question mostly concern a theoretical
aspect rather than practical one. To be not misunderstood, what is
the mathematical model that one should apply to be able to deal
with twinned intensities with their errors? I mean, I+_what? I ask
this In order to state some general consideration on the accuracy
about the recovery the true intensities on varying of alpha. Thanks
Fulvio
Fulvio Saccoccia PhD Dept. of Biochemical Sciences Sapienza
University of Rome 5, Piazzale A. Moro 00185 phone +39 0649910556
Messaggio Originale Da: herman.schreu...@sanofi.com
Inviato: 06/11/2013, 17:25 A: CCP4BB@JISCMAIL.AC.UK Oggetto:
[ccp4bb] AW: [ccp4bb] uncertainites associated with intensities
from twinned crystals
Dear Fulvio, you cannot detwin perfectly twinned data with this
formula. The term (1-2α) becomes zero, so you are dividing by zero.
With good refinement programs (ShelX, Refmac), refinement is done
against twinned data, which is better than to detwin the data with
the formula you mention.
As I understand it, to get map coefficients, the calculated
contribution of the twin domain (Fcalc’s) is substracted from Fobs
(with the appropriate weighting factors), so what you see in coot
is detwinned electron density. In practical terms, the only thing
you have to do is to specify the TWIN keyword in Refmac.
Best regards, Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag
von Fulvio Saccoccia Gesendet: Mittwoch, 6. November 2013 16:58 An:
CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] uncertainites associated
with intensities from twinned crystals
Dear ccp4 users
a question about the recovering of true intensities from merohedral
twinned crystal. Providing alpha and the twin operator one should
be able to recover the intensities from the formulas:
I(h1) =
Re: [ccp4bb] R: [ccp4bb] AW: [ccp4bb] uncertainites associated with intensities from twinned crystals; Sorry for HTML.
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear Jens,
thanks for setting this right.
Best,
Tim
On 11/07/2013 07:53 AM, Jens Kaiser wrote:
Fulvio, Tim, error propagation is correct, but wrongly applied in
Tim's example. s_f= \sqrt{ \left(\frac{\partial f}{\partial {x}
}\right)^2 s_x^2 + \left(\frac{\partial f}{\partial {y} }\right)^2
s_y^2 + \left(\frac{\partial f}{\partial {z} }\right)^2 s_z^2 +
...} (see
http://en.wikipedia.org/wiki/Propagation_of_uncertainty#Simplification)
The uncertainty in a derived magnitude is always larger than any
individual uncertainty, so no subtraction, anytime. Furthermore,
in Tim's example you could end up with negative sigmas..
HTH,
Jens
On Thu, 2013-11-07 at 04:44 +0100, Tim Gruene wrote:
Dear Fulvio,
with simple error propagation, the error would be sigma(I(h1)) =
(1-α)sigma(Iobs(h1))-α*sigma(Iobs(h2))/(1-2α)
would it not?
Although especially for theoretical aspects you should be
concerned about division by zero.
Best, Tim
On 11/06/2013 05:54 PM, Fulvio Saccoccia wrote:
Thank you for reply. My question mostly concern a theoretical
aspect rather than practical one. To be not misunderstood, what
is the mathematical model that one should apply to be able to
deal with twinned intensities with their errors? I mean,
I+_what? I ask this In order to state some general
consideration on the accuracy about the recovery the true
intensities on varying of alpha. Thanks Fulvio
Fulvio Saccoccia PhD Dept. of Biochemical Sciences Sapienza
University of Rome 5, Piazzale A. Moro 00185 phone +39
0649910556
Messaggio Originale Da: herman.schreu...@sanofi.com
Inviato: 06/11/2013, 17:25 A: CCP4BB@JISCMAIL.AC.UK Oggetto:
[ccp4bb] AW: [ccp4bb] uncertainites associated with
intensities from twinned crystals
Dear Fulvio, you cannot detwin perfectly twinned data with
this formula. The term (1-2α) becomes zero, so you are dividing
by zero. With good refinement programs (ShelX, Refmac),
refinement is done against twinned data, which is better than
to detwin the data with the formula you mention.
As I understand it, to get map coefficients, the calculated
contribution of the twin domain (Fcalc’s) is substracted from
Fobs (with the appropriate weighting factors), so what you see
in coot is detwinned electron density. In practical terms, the
only thing you have to do is to specify the TWIN keyword in
Refmac.
Best regards, Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im
Auftrag von Fulvio Saccoccia Gesendet: Mittwoch, 6. November
2013 16:58 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb]
uncertainites associated with intensities from twinned
crystals
Dear ccp4 users
a question about the recovering of true intensities from
merohedral twinned crystal. Providing alpha and the twin
operator one should be able to recover the intensities from the
formulas:
I(h1) = (1-α)Iobs(h1)-αIobs(h2)/(1-2α)
I(h2) = -αIobs(h1)+(1+α)Iobs(h2)/(1-2α)
as stated in many papers and books*.
However I was wondering about the uncertainties associated to
these measurements, I mean: for all physical observable an
uncertainty should be given.
Hence, what is the uncertainty associated to a perfect
merohedrally twinned crystal (alpha=0.5)? It is clear that in
this case we drop in a singular value of the above formulas.
Please, let me know your hints or your concerns on the matter.
Probably there is something that it is not so clear to me.
Thanks in advance
Fulvio
ref. **(C. Giacovazzo, H. L. Monaco, G. Artioli, D. Viterbo,
M. Milaneso, G. Ferraris, G. Gilli, P. Gilli, G. Zanotti and M.
Catti. Fundamentals of Crystallography, 3rd edition. IUCr Texts
on Crystallography No. 15, IUCr/Oxford University Press, 2011;
Chandra, N., Acharya, K. R., Moody, P. C. (1999). Acta Cryst.
D55. 1750-1758)
--
Fulvio Saccoccia, PhD
Dept. of Biochemical Sciences A. Rossi Fanelli
Sapienza University of Rome
Tel. +39 0649910556
- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
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Re: [ccp4bb] [ccp4bb] AW: [ccp4bb] uncertainites associated with intensities from twinned crystals; Sorry for HTML.
Fulvio,
First, to your point 2): Iobs(h1) and Iobs(h2) as well as Itrue(h1)
and Itrue(h2) are /not/ correlated! The Iobses are /related/ to the
Itrues by alpha (and the twin law), but the Itrues are totally
uncorrelated to each other, and so are the Iobses, in my opinion (even
though those will become more and more equal as alpha approaches 0.5,
but this is not a correlation! And at alpha = 0.5 this formalism breaks
down, anyways). So I do think that the simple error propagation is valid
here.
Now for your point 1): The formula I gave is only valid if you have an
analytical relationship between the magnitudes you measure and the
magnitudes you extract (and no correlation between them). For
non-merohedral twins, this is not true, as you'll have to make that
decision on a reflection by reflection base, so this is definitely /not/
generally applicable in that situation.
And yes, the uncertainties associated with /detwinned/ intensities are
much larger than the uncertainties associated with your measured data.
This is one (but not the most important) reason, to refine against
intensities and make the twin law part of your model.
Hope that makes sense,
Jens
On Thu, 2013-11-07 at 09:22 +0100, fulvio.saccoc...@uniroma1.it wrote:
Dear all,
thank you for your reply. I would summarize my concerns and opinions,
so
far:
1) for QTLS (non-merohedral twinning - non intersecting lattices) I think one
should consider the variables as independent and random and it is possible to
recover the true intensities of a unique lattice from the stronger
diffracting
one (see for example Jenni Ban, 2009, Acta D65, 101-111). Hence, the
quadratic formula (reported fomr Jens Kaiser) can be applied;
2) for TLS (merohedral twinning - perfectly overlapping spots) I think one
should not consider the two variable independent since they are related by
alpha (see the formulas I reported in my first message). In this case, I
think
the right formula should be that reported by Tim Grune, that as far as I know
overestimates the true error but in this case the quadratic is not applicable.
Therefore, one would be prone to conclude that the uncertainties associated
to
merohedral-twinned crystals are higher than regular crystals or
non-merohedral
crystals. What's your opinion about?
In data mercoledì 6 novembre 2013 23:29:01, Jens Kaiser ha scritto:
Tassos,
I'm no expert either, and there are caveats for using this formula on
correlated magnitudes. But I would assume that the intensities of twin
related reflections should be independent from each other (that's my
understanding of the sigmoid cumulative intensity distribution of
twins). Thus, I think the simple Gaussian error propagation should be
applicable to uncertainty estimates in detwinned intensities.
Cheers,
Jens
On Thu, 2013-11-07 at 08:09 +0100, Anastassis Perrakis wrote:
Dear Jens,
That formula for error propagation is correct for independent
measurements.
Does this assumption stand true for Intensities in twinning? I am no
expert, but I would think not.
Tassos
On 7 Nov 2013, at 7:53, Jens Kaiser wrote:
Fulvio, Tim,
error propagation is correct, but wrongly applied in Tim's
example.
s_f= \sqrt{ \left(\frac{\partial f}{\partial {x} }\right)^2 s_x^2 +
\left(\frac{\partial f}{\partial {y} }\right)^2 s_y^2 +
\left(\frac{\partial f}{\partial {z} }\right)^2 s_z^2 + ...} (see
http://en.wikipedia.org/wiki/Propagation_of_uncertainty#Simplification)
The uncertainty in a derived magnitude is always larger than any
individual uncertainty, so no subtraction, anytime. Furthermore, in
Tim's example you could end up with negative sigmas..
HTH,
Jens
On Thu, 2013-11-07 at 04:44 +0100, Tim Gruene wrote:
Dear Fulvio,
with simple error propagation, the error would be
sigma(I(h1)) = (1-α)sigma(Iobs(h1))-α*sigma(Iobs(h2))/(1-2α)
would it not?
Although especially for theoretical aspects you should be concerned
about division by zero.
Best,
Tim
On 11/06/2013 05:54 PM, Fulvio Saccoccia wrote:
Thank you for reply. My question mostly concern a theoretical
aspect rather than practical one. To be not misunderstood, what is
the mathematical model that one should apply to be able to deal
with twinned intensities with their errors? I mean, I+_what? I ask
this In order to state some general consideration on the accuracy
about the recovery the true intensities on varying of alpha. Thanks
Fulvio
Fulvio Saccoccia PhD Dept. of Biochemical Sciences Sapienza
University of Rome 5, Piazzale A. Moro 00185 phone +39 0649910556
Messaggio Originale Da: herman.schreu...@sanofi.com
Inviato: 06/11/2013, 17:25 A: CCP4BB@JISCMAIL.AC.UK Oggetto:
[ccp4bb] Joint CCP4-6.4.0 and ARP/wARP-7.4 Update
Dear CCP4 Users, CCP4 Core Team would like to apologise for temporary withdrawal of update 6.4.0-002, announced earlier this week. The withdrawal is due to a technical fault in the update mechanism, which will be rectified soon. Currently, the update is put on hold and is not visible for client installations. A few users reported problems with starting ccp4i immediately after installation of the update. Should you have experienced the same, please start ccp4um from terminal: sudo /path-to/ccp4-6.4.0/bin/ccp4um and uninstall update 6.4.0-002 by unchecking the corresponding box and pressing Apply (use 'sudo' *only* if your ccp4 is installed in write-protected area). After that, everything should be back normal. Windows users, as well as those who are able to run ccp4i after 2nd update, need do nothing. I would like to acknowledge prompt bug reports from responsible users on this occasion, which helped us to minimise the negative effect of the bug on all others. CCP4 as a project depends on the feedback from user community, and Core Team is grateful for all the help and feedback we receive. Please refrain from update attempts until further announcement or until new updates are reported available by your ccp4i. Many thanks, Eugene Krissinel Begin forwarded message: From: Andrey Lebedev andrey.lebe...@stfc.ac.ukmailto:andrey.lebe...@stfc.ac.uk Date: 6 November 2013 12:52:31 GMT To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Joint CCP4-6.4.0 and ARP/wARP-7.4 Update Reply-To: andrey.lebe...@stfc.ac.ukmailto:andrey.lebe...@stfc.ac.uk Dear CCP4 Users An update for the CCP4-6.4.0 series has just been released. This is the first update for both CCP4 and ARP/wARP in tandem. The ARP/wARP component of this update will be available to those Mac and Linux users who have previously installed both packages via the CCP4 Package manager or from the joint bundle downloaded from CCP4 web-site. A standalone ARP/wARP package including the current update can be downloaded from the EMBL-Hamburg site at www.arp-warp.orghttp://www.arp-warp.org/. The following changes will be applied to the ARP/wARP installation: • The use of non-crystallographic symmetry for protein chain tracing has been enabled (it was accidentally disabled in version 7.4). • The `SAD refinement protocol' (Murshudov et al 2011) has undergone many fixes and now works well. • The routine for ligand identification has been made faster and some small bugs have been fixed. • A few bugs have been fixed in the ARP/wARP installer and shell-specific problems sometimes occurring on Linux should now be cured. • The manual has been updated. The following changes will be applied to the CCP4-6.4.0 installation: • MrBUMP (all): a new option of model re-building after SHELXE; a fix for phaser_sculptor dispatcher. • Xia2 (windows): update to release 0.3.6.3 (fixes ctruncate issue). • aimless (all): bug fixes. • pointless (all): bug fixes. • ViewHKL (all): additional visual control to enhance weak reflections and bug fixes. • truncate (Mac): Fixed harvesting option of old truncate. • Molrep: New interface. • Update (all): Increased maximum waiting time for background update check in ccp4i. Andrey Lebedev -- Scanned by iCritical. -- Scanned by iCritical.
[ccp4bb] few questions about resolving new structure through MR
Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
Count yourself lucky that you may have a partial solution for your structure with only 30% identity. The question now is: can you see any reasonable, traceable electron density for domain A? Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/7/2013 11:36 AM, Zhihong Yu wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - Original Message - From: Zhihong Yu nkyuz...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, November 7, 2013 11:36:51 AM Subject: [ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] SA-omit map
Would an omit map calculated after removing the ssDNA from the model and then
gently randomizing the remaining coodinates using the NOISE option in pdbset
(possibly not in the GUI version)
NOISE [maximum_shift] [subkeys]
Introduce random shifts into atom positions in orthogonal coordinates.
maximum_shift maximum shift (Angs)
defaults to 0.2 Angs, fails if greater than 0.5 Angs
be a possible alternative to a simulated annealing technique? Would it not be
similarly effective at removing any memory bias in the remaining coodinates,
and very easy to do?
Having said that, it is usually simplest to do what the referee requests, if at
all possible. And if your DNA isn't there in the omit map, then you have no
reason to suggest that it is there at all.
best wishes
Pete
On 4 Nov 2013, at 18:02, Mark Brooks mark.x.bro...@gmail.com wrote:
Maybe try CNS or SFCheck:
http://groups.yahoo.com/neo/groups/cnsbb/conversations/messages/1720
To improve Phenix maps, maybe try increasing the number of boxes (the
parameter IIRC n_box_target= )
http://www.phenix-online.org/documentation/autobuild.htm
In CNS, you can decrease the starting temperature in the annealing section,
to reduce the 'violence' of the simulated annealing:
http://cns-online.org/cgi-bin/cns_solve_1.2/cns_view.cgi?file=inputs/xtal_refine/composite_omit_map.inp
---8---Snip--8--
{* starting temperature *}
{===} temperature=500;
---8---Snip--8--
As said elsewhere, if your map is still poor, maybe it's trying to tell you
something...
Mark
On 4 November 2013 06:36, dengzq1987 dengzq1...@gmail.com wrote:
Dear all,
Recently, I received the comments from referees, they asked for the SA-omit
map of the ssDNA of our protein-DNA complex. They said that simulated
annealing omit map better than a biased 2Fo-Fc. The ssDNA consists of seven
thymidine nucleotide. Our data diffracted to 2.65A，but the data quality is
not good and twin. We tried to produce SA-omit map using phenix. The map is
really bad. Does anyone have suggestion to refine the map? Thank you!
Bests,
zq Deng
2013-11-04
dengzq1987
Prof Peter Artymiuk
Krebs Institute
Department of Molecular Biology Biotechnology
University of Sheffield
Sheffield
S10 2TN
ENGLAND
Re: [ccp4bb] few questions about resolving new structure through MR
First of all, thanks so much for your reply. To Roger: NO, unfortunately I cannot see too much traceable electron density outside the placed atoms, so I think just as Greg said, it's only a model-biased solution. To Greg: YES, I also realized that the input model should be very important, so I'm going to try only backbone of structureX, or build a homology model of domainB first and then put it as a search model. Actually, I asked those questions because I had no idea that even I can correctly place domainB using structureX as a search model, can I really resolve the full length structure? after all, the resolution is only 3.5, and the domainB is only contain 40% residues of the full length. I really want to get some opinions from you expert whether it's worth to spend much time on trying to resolve the strucutre through MR based on current dataset. Or I have to prepare SeMet protein to get experimental phasing information? Thank you all again and look forward to hearing from more expert! Zhihong On Thu, Nov 7, 2013 at 1:25 PM, Greg Costakes gcost...@purdue.edu wrote: The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -- *From: *Zhihong Yu nkyuz...@gmail.com *To: *CCP4BB@JISCMAIL.AC.UK *Sent: *Thursday, November 7, 2013 11:36:51 AM *Subject: *[ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Zhihong, in many labs a SeMet prep is not much effort and can be done within a week or two, if you express in E.coli. Unless the costs are a limiting factor I would certainly go this way. However, with native data to 3.5A I suggest you contact somebody knowledgeable to help you collect a MAD data set - this will be difficult enough. While the protein is being expressed and purified you can start a couple of MR jobs with different search models - I would use sculptor or mrtailor, though, instead of a plain poly-ALA model: don't make life more complicated than necessary. Regards, Tim On 11/07/2013 08:31 PM, Zhihong Yu wrote: First of all, thanks so much for your reply. To Roger: NO, unfortunately I cannot see too much traceable electron density outside the placed atoms, so I think just as Greg said, it's only a model-biased solution. To Greg: YES, I also realized that the input model should be very important, so I'm going to try only backbone of structureX, or build a homology model of domainB first and then put it as a search model. Actually, I asked those questions because I had no idea that even I can correctly place domainB using structureX as a search model, can I really resolve the full length structure? after all, the resolution is only 3.5, and the domainB is only contain 40% residues of the full length. I really want to get some opinions from you expert whether it's worth to spend much time on trying to resolve the strucutre through MR based on current dataset. Or I have to prepare SeMet protein to get experimental phasing information? Thank you all again and look forward to hearing from more expert! Zhihong On Thu, Nov 7, 2013 at 1:25 PM, Greg Costakes gcost...@purdue.edu wrote: The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - -- *From: *Zhihong Yu nkyuz...@gmail.com *To: *CCP4BB@JISCMAIL.AC.UK *Sent: *Thursday, November 7, 2013 11:36:51 AM *Subject: *[ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.15 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFSe/udUxlJ7aRr7hoRAqMvAJ9Pl9KKQL1Ce56HrNe7wKo+EO2U1gCg4rLF /FqvLWRYfxM3/3MJjX5HyPQ= =RNgU -END PGP SIGNATURE-
Re: [ccp4bb] few questions about resolving new structure through MR
Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] few questions about resolving new structure through MR
Thanks Francis, No, only one molecule in the asu. The Matthews Coefficient is 3.3, corresponding solvent content is 62.6%, maybe that's why this crystal show such weak diffraction? Zhihong On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.eduwrote: Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Fwd: Citing Aimless
Dear all I am also looking for aimless reference. Which paper should I cite? Thanks for the powerful tools for the community. Best, Joe On Sun, Feb 24, 2013 at 9:58 PM, Morten Groftehauge m...@mb.au.dk wrote: Hi guys, In the log from Aimless the only reference mentioned is the 1994 CCP4 paper and then as well as any specific reference in the program write-up. First of all, is this the correct CCP4 reference to use? And second of all, since running Aimless through the interface always invokes Pointless and ctruncate, wouldn't I always cite those as well? I might not need Pointless to determine the space group but it doesn't hurt and doesn't Aimless use information from that run? Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University -- Morten K Grøftehauge, PhD Pohl Group Durham University
Re: [ccp4bb] few questions about resolving new structure through MR
Dear Debanu, Thanks for your detailed reply. The Z-Score in my current MR trial is only 4.2, which means that domainB was not correctly placed at all, the observed density is indeed model biased density. Since it's my first experience of resolving a new structure, I'm really not sure whether it's worth to put too much efforts on MR based on current 3.5A dataset and only a structure with low homology with one domain. From your reply, I think it's still worth to try a little bit and got information as much as I can. I'm going to try MR Rosetta first. Best Regards! Zhihong On Thu, Nov 7, 2013 at 6:36 PM, Das, Debanu deb...@slac.stanford.eduwrote: Hi Zhihong, The 3.5A diffraction could be due to many reasons: N- and C-term regions, interdomain linker possibly giving rise to molecular flexibility, quality of the particular crystals, cryo, purification, tags, etc. One thing to try is to run secondary structure predictions (or BLAST against PDB, FFAS) on the N- and C-term regions and optimize your construct to exclude some or all of them, especially if you have evidence that they might not be functionally important. 1) Observing density corresponding to your protein sounds promising. What is your PHASER Z-score? Usually Z-scores 8 are indicative of correct solutions so if you are confident that you have the correct placement/solution for domain B, you can try to optimize refinement/model using DEN or MR Rosetta or morph_model. 2) Try the above and see if you can improve your model/maps/R-values. Try optimizing your model (changing residues, removing loops, etc.) by homology modeling (you can try using the PSI Modeling Portal http://www.proteinmodelportal.org/) or other similar services or try different programs individually. In addition, try to obtain a homology model of domainA (including model building with Rosetta/Robetta). Additional phasing information by experimental phasing using SeMet or heavy atoms will be best, but is often easier said than done. Since you are at the MR stage, it will be useful if you can squeeze as much information as you can from MR efforts. If you are sure you have domainB placed correctly (and can also obtain a reliable solution for domainA), your MR phases can be used later on to locate heavy atom sites by difference Fourier methods and you can also combine with experimental phases in non-optimal cases Best, Debanu. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhihong Yu [nkyuz...@gmail.com] Sent: Thursday, November 07, 2013 2:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] few questions about resolving new structure through MR Thanks Francis, No, only one molecule in the asu. The Matthews Coefficient is 3.3, corresponding solvent content is 62.6%, maybe that's why this crystal show such weak diffraction? Zhihong On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.edu mailto:francis.re...@colorado.edu wrote: Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.commailto: nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Fwd: Citing Aimless
It should be printed at the end of the log file P.R.Evans and G.N.Murshudov, 'How good are my data and what is the resolution?' Acta Cryst. D69, 1204-1214 (2013). Phil On 8 Nov 2013, at 11:35, Zheng Zhou zhengzho...@gmail.com wrote: Dear all I am also looking for aimless reference. Which paper should I cite? Thanks for the powerful tools for the community. Best, Joe On Sun, Feb 24, 2013 at 9:58 PM, Morten Groftehauge m...@mb.au.dk wrote: Hi guys, In the log from Aimless the only reference mentioned is the 1994 CCP4 paper and then as well as any specific reference in the program write-up. First of all, is this the correct CCP4 reference to use? And second of all, since running Aimless through the interface always invokes Pointless and ctruncate, wouldn't I always cite those as well? I might not need Pointless to determine the space group but it doesn't hurt and doesn't Aimless use information from that run? Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University -- Morten K Grøftehauge, PhD Pohl Group Durham University
