Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

As you suggest, it depends on the contour level. Looking through a list of 17 
dicarboxylates that I found problematic, there were 4 (actually two and their 
ncs-mates) that showed disconnected density for the carboxylate at 1.4 sigma as 
in the figure I sent. Going up to 2 sigma, four more became disconnected and 
linear (backbone density is continuous to 3.5 sigma). Going down to 0.6 sigma, 
disconnected residual density showed up for several more. So I guess there are 
varying degrees of decarboxylation, and varying extent of retention of the 
fragment.  I have the impression these are mostly on the protein/solvent 
boundary, which could explain their disorder, but perhaps would also expose 
them to greater concentration of radicals generated in the solvent channels(?).

Ed

On 05/04/2017 06:25 AM, Andrew Leslie wrote:

Dear Ed,

   I find your electron density quite interesting, because 
generally (I think, I would be happy to be corrected on this) when 
de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
evidence of what happens to the resulting CO2 group. One interpretation of this 
is that it diffuses away from the side chain and is effectively totally 
disordered, so no electron density is seen, but I was surprised that this would 
always be the case, especially as I would have thought that diffusion would be 
quite limited at 100K (maybe I’m wrong about that too, but that is supposed to 
be one reason why radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew



On 3 May 2017, at 22:19, Edward A. Berry  wrote:



On 05/03/2017 02:46 PM, Gerard Bricogne wrote:

Dear Ed,

  Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

  Gerard.


Actually some of them do appear decarboxylated. Is that something that can 
happen? In the crystal, or as radiation damage?
However when there is density for the carboxylate (figure), it appears 
continuous and linear, doesn't break up into spheres at H-bonding distance - 
almost like the CO2 is still sitting there- but I guess it would get hydrated 
to bicarbonate. I could use azide. Or maybe waters with some disorder.
Thanks,
eab

Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
comparison, not part of the model.

[ccp4bb] Postdoc position available - Imperial College London - Prof Dale Wigley

[Wilkinson, Martin]

——

Martin Wilkinson
mwilk...@ic.ac.uk

Imperial College
Lab 245
2nd Floor, SAF building
Imperial College Road
London
SW7 2AZ

——



HM2017001 Research Associate Advert QC SW - extended2.pdf
Description: HM2017001 Research Associate Advert QC SW - extended2.pdf

Re: [ccp4bb] Poor density fit.

[Vipul Panchal]

Thanks Bert,

I did understand not to delete such atoms or set occupancy to zero from
forum and literature. However, colleagues in the vicinity informed to take
approaches i mentioned earlier. Therefore, I thought to take opinion from
forum if something has changed recently.

Thanking,



On Thu, May 4, 2017 at 8:55 PM, Bert Van-Den-Berg <
bert.van-den-b...@newcastle.ac.uk> wrote:

> This has been discussed before, I guess more than once
>
> I think most people (I'm sure i'll be corrected if wrong) would favor not
> removing any atoms or setting occupancies to zero and let the invisible
> atoms be accounted for by high B-factors (either set manually or just
> letting refinement do its thing).
>
>
> Bert
>
>
> --
> *From:* CCP4 bulletin board  on behalf of Vipul
> Panchal 
> *Sent:* 04 May 2017 16:12
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Poor density fit.
>
> HI all.
>
> I am solving protein structure with 2.16A resolution. There are two chain
> in an asymmetric unit. I see that in one of the chain, many residues'
> density for side chains is incomplete and therefore results in poor density
> fit.
>
> *I want to know your opinions for the approach I have taken. Figures
> relevant to each approach have been attached herewith.*
>
> *Case1*: There is no experimental density at all. Therefore, i have
> deleted side chains to Gly.
> *Case2*: Though there is incomplete density for Leu, it is enough to
> suggest its rotamer. In this case, as may be seen, i have just set
> occupancy for atoms without density(CG, CD1, CD2) to zero.
>
> Hopeful for the response.
>
> --
> Vipul Panchal
> Senior Research Fellow,
> Respiratory disease and biology,
> CSIR-IGIB
> (M)-9540113372
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] AW: [ccp4bb] Poor density fit.

[Herman . Schreuder]

Dear Vipul,

the first thing I would check is why one chain has good and the other chain has 
poor side chain density. Are the B-factors of one chain much higher than of the 
other? Does one chain have more/better crystal contacts to stabilize its 
position in the crystal? Is the structure well-refined (e.g. R~20%; Rfree~25%)?

If this has all been checked, I would do as Bert suggested and leave the side 
chains intact and let the B-factors take care of the disorder. However, this is 
a very contentious issue, with probably 50% of the board members in favor of 
leaving the side chains intact and the other 50% in favor of truncating 
undefined side chains. Both approaches have their merits and I would do what 
you personally feel is the best, rather than setting off another mega-thread on 
this subject. You could try to google previous threads on this issue if you are 
interested! ;-)

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul 
Panchal
Gesendet: Donnerstag, 4. Mai 2017 17:13
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Poor density fit.

HI all.

I am solving protein structure with 2.16A resolution. There are two chain in an 
asymmetric unit. I see that in one of the chain, many residues' density for 
side chains is incomplete and therefore results in poor density fit.

I want to know your opinions for the approach I have taken. Figures relevant to 
each approach have been attached herewith.

Case1: There is no experimental density at all. Therefore, i have deleted side 
chains to Gly.
Case2: Though there is incomplete density for Leu, it is enough to suggest its 
rotamer. In this case, as may be seen, i have just set occupancy for atoms 
without density(CG, CD1, CD2) to zero.

Hopeful for the response.

--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Poor density fit.

[Bert Van-Den-Berg]

This has been discussed before, I guess more than once

I think most people (I'm sure i'll be corrected if wrong) would favor not 
removing any atoms or setting occupancies to zero and let the invisible atoms 
be accounted for by high B-factors (either set manually or just letting 
refinement do its thing).


Bert



From: CCP4 bulletin board  on behalf of Vipul Panchal 

Sent: 04 May 2017 16:12
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Poor density fit.

HI all.

I am solving protein structure with 2.16A resolution. There are two chain in an 
asymmetric unit. I see that in one of the chain, many residues' density for 
side chains is incomplete and therefore results in poor density fit.

I want to know your opinions for the approach I have taken. Figures relevant to 
each approach have been attached herewith.

Case1: There is no experimental density at all. Therefore, i have deleted side 
chains to Gly.
Case2: Though there is incomplete density for Leu, it is enough to suggest its 
rotamer. In this case, as may be seen, i have just set occupancy for atoms 
without density(CG, CD1, CD2) to zero.

Hopeful for the response.

--
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] Poor density fit.

[Vipul Panchal]

HI all.

I am solving protein structure with 2.16A resolution. There are two chain
in an asymmetric unit. I see that in one of the chain, many residues'
density for side chains is incomplete and therefore results in poor density
fit.

*I want to know your opinions for the approach I have taken. Figures
relevant to each approach have been attached herewith.*

*Case1*: There is no experimental density at all. Therefore, i have deleted
side chains to Gly.
*Case2*: Though there is incomplete density for Leu, it is enough to
suggest its rotamer. In this case, as may be seen, i have just set
occupancy for atoms without density(CG, CD1, CD2) to zero.

Hopeful for the response.

-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] very high B-factor

[yanqiaoling2782048]

Sorry, the R-factor gap of no6 is 5.23%, not 6.23%, I have made a mistake.

I have check the b-factor of the residue with "B-factor variance" value of 417 
in coot-->menu-->distance-->residue info., the b-factor value of each atom of 
the residue is very similar with that of in pdb file, though not exactly the 
same. In addition, among the residues with high "B-factor variance" , some are 
fitting quite well into the density, but some have very poor density for side 
chain. It seems that  "B-factor variance" and b-factor doesn't mean the same 
thing. I still don't know what's the "B-factor variance" stands for?
Moreover, if density is missing for residue side, I would like to pick an 
appropriate rotamer and let the b-factors rise to account for disorder.
I really appreciate your advice.



At 2017-05-04 19:45:14, "Vipul Panchal"  wrote:

1) the difference is 5.23 % and it is good and acceptable. 
2) if higher value belongs to residue, then you need to change the rotamer 
Using coot ,  follow menu-->distance-->residue info. You will get to know to 
whom 417 B factor value belongs. Meanwhile, just check if modeled residue or 
part of residue has relevant density. If density is missing for part of 
residue, then set occupancy to zero for the relevant atom(s). If density is 
missing for the model of residue, then just remove side chain. I would 
recommend to check density fit (coot-->menu--> validate--> density fit) for all 
residues with >50 B factor value (coot-->menu--> validate--> temp. fact. 
variance analysis). 




On 04-May-2017 3:26 PM, "yanqiaoling2782048"  wrote:

Hi Vipul,


Thanks for your quick reply.


1. Actually, i mainly comparing the gap between Rwork and Rfree which should be 
<5%, and then the overall b-factor, RMSD of bond and angle. For me, no5 is the 
best one, but the "B factor variance Graphs" make me uncomfortable. The 
R-factor gap of no6 is 6.23%, does this mean overfitting?


2. The b-factor value of 76 is belong to the atom of the residue obtained from 
the pdb file. I'm not sure whether the B-factor in  "B factor variance Graphs" 
belong to residue or atom, but i guess 417 belongs to residue. What's the 
relationship of the two factor?  


Best regards,
Qiaoling Yan 




At 2017-05-04 15:58:36, "Vipul Panchal"  wrote:

Hi,


1>
Well, it seems you are just comparing Max value across all protocols. You 
should compare average values as it also takes into account no. of atoms with 
given values of B factor. As per refinement results, it seems though no. 6 has 
highest B factor value, number of atomic outliers are relatively low. So if you 
ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you should 
go ahead with no.6 is strategy.


2>
When comparing B factor values of 417 vs 76, you should consider what it 
belongs to. As i may guess, 417 value belongs to an atom of residue whereas 76 
belongs to residue. Meaning 417 is individual B factor whereas 76 is grouped B 
factor.




On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048  
wrote:

Dear all,

I'm working on a crystal structure with resolution of 2.2A. At the final step, 
I use different strategies to refine the structure, they are:
no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)

no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 / 
optimize_xyz/adp_weight=true
results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/max/average)

no6: strategy=individual_sites+individual_adp / anisotropic for all residues 
and isotropic for water /  
 set_b_iso=10 / optimize_xyz/adp_weight=true
results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7 (min/max/average)
PS: the results is read from pdb file

The results showed that the strategy of no5 is the best one. But the "B factor 
variance Graphs" generated by coot with menu/validate/temp.fact.variance 
analysis, have shown that no6 have the lowest B-factors (the attached figure is 
the B-factor graphs of three pdb files). And my questions are:
1. Which strategy should I choose to refine my structure? Or any other 
suggestions to refine the structure at 2.2A resolution?
2. Does it possible that some residues have very high B-factor in "B factor 
variance Graphs", while in the pdb file， the b-factor of corresponding residues 
are relatively low? For example, one residue have B-factors of 417 in "B factor 
variance Graphs", but in PDB file the b factor is 76. Does the two factor mean 
the same thing?
3. If i want to set the weight manually, which parameter should i set, 
wxc/wxc_scale? or others?

Thanks in advance.

Best regards,
Qiaoling Yan






 






--

Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372




 








 

Re: [ccp4bb] very high B-factor

[Pavel Afonine]

Hi,


I'm working on a crystal structure with resolution of 2.2A. At the final
> step, I use different strategies to refine the structure, they are:
> no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
> results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)
>
> no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 /
> optimize_xyz/adp_weight=true
> results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/
> max/average)
>


make sense to me, both - the strategy and results. Getting the same results
using different starting B is also a good sign.



> no6: strategy=individual_sites+individual_adp / anisotropic for all
> residues and isotropic for water /
>  set_b_iso=10 / optimize_xyz/adp_weight=true
> results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7
> (min/max/average)
> PS: the results is read from pdb file
>

This does not make sense: 2.2A isn't good enough to refine all residues
with anisotropic ADP.



> The results showed that the strategy of no5 is the best one.
>


Both, no4 and no5 looks same to me.



> And my questions are:
> 1. Which strategy should I choose to refine my structure? Or any other
> suggestions to refine the structure at 2.2A resolution?
>


Your no4 and no5 look fine. Make sure you let phenix.refine to add water
automatically as part of refinement run. Check manually at the very final
stage.



> 2. Does it possible that some residues have very high B-factor in "B
> factor variance Graphs", while in the pdb file， the b-factor of
> corresponding residues are relatively low? For example, one residue have
> B-factors of 417 in "B factor variance Graphs", but in PDB file the b
> factor is 76. Does the two factor mean the same thing?
>


I don't know how that analysis works. Perhaps it's looking at variance of
local Bs not the absolute value.



> 3. If i want to set the weight manually, which parameter should i set,
> wxc/wxc_scale? or others?
>

wxc_scale for coordinates, wxu_scale for ADP. Normally, though, you are not
expected to do this if you let the program to optimize weights.

Pavel

Re: [ccp4bb] very high B-factor

[Robbie Joosten]

Hi Vipul and Qiaoling,

Setting occupancies to 0 will give somewhat misleading results. It is good to 
test whether density will disappear or not, but not for the final model. Users 
of the model might misinterpret it. Especially if you start leaving out 
'random' poorly fitting atoms.

High B-factors can occur and the average seems fine. High variance can be an 
indication of too loose B-factor restraints. Since you asked for suggestions of 
other protocols on the CCP4bb: Have you tried Refmac? And a shameless plug: 
Have you tried pdb-redo? Among other things, this tries to find a good protocol 
to get the best performance out of Refmac.

Cheers,
Robbie

> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Vipul Panchal
> Sent: Thursday, May 04, 2017 13:45
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] very high B-factor
> 
> 1) the difference is 5.23 % and it is good and acceptable.
> 2) if higher value belongs to residue, then you need to change the rotamer
> Using coot ,  follow menu-->distance-->residue info. You will get to know to
> whom 417 B factor value belongs. Meanwhile, just check if modeled residue
> or part of residue has relevant density. If density is missing for part of
> residue, then set occupancy to zero for the relevant atom(s). If density is
> missing for the model of residue, then just remove side chain. I would
> recommend to check density fit (coot-->menu--> validate--> density fit) for
> all residues with >50 B factor value (coot-->menu--> validate--> temp. fact.
> variance analysis).
> 
> 
> On 04-May-2017 3:26 PM, "yanqiaoling2782048"
>  wrote:
> 
> 
>   Hi Vipul,
> 
>   Thanks for your quick reply.
> 
>   1. Actually, i mainly comparing the gap between Rwork and Rfree
> which should be <5%, and then the overall b-factor, RMSD of bond and
> angle. For me, no5 is the best one, but the "B factor variance Graphs" make
> me uncomfortable. The R-factor gap of no6 is 6.23%, does this mean
> overfitting?
> 
>   2. The b-factor value of 76 is belong to the atom of the residue
> obtained from the pdb file. I'm not sure whether the B-factor in  "B factor
> variance Graphs" belong to residue or atom, but i guess 417 belongs to
> residue. What's the relationship of the two factor?
> 
> 
>   Best regards,
>   Qiaoling Yan
> 
> 
> 
> 
>   At 2017-05-04 15:58:36, "Vipul Panchal" 
> wrote:
> 
> 
>   Hi,
> 
>   1>
>   Well, it seems you are just comparing Max value across all
> protocols. You should compare average values as it also takes into account
> no. of atoms with given values of B factor. As per refinement results, it
> seems though no. 6 has highest B factor value, number of atomic outliers are
> relatively low. So if you ask me no. 6 is the best one.
>   Coot results are also in congruent with refinement results. I
> think you should go ahead with no.6 is strategy.
> 
>   2>
>   When comparing B factor values of 417 vs 76, you should
> consider what it belongs to. As i may guess, 417 value belongs to an atom of
> residue whereas 76 belongs to residue. Meaning 417 is individual B factor
> whereas 76 is grouped B factor.
> 
> 
>   On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048
>  wrote:
> 
> 
>   Dear all,
> 
>   I'm working on a crystal structure with resolution of
> 2.2A. At the final step, I use different strategies to refine the structure, 
> they
> are:
>   no4: strategy=individual_sites+individual_adp+tls /
> set_b_iso=20
>   results: Rwork/free=0.2052/0.2658  b-
> factor=11.4/136.8/48(min/max/average)
> 
>   no5: strategy=individual_sites+individual_adp+tls /
> set_b_iso=10 / optimize_xyz/adp_weight=true
>   results: Rwork/free=0.2161/0.2639  b-
> factor=11.3/135.2/48.4(min/max/average)
> 
>   no6: strategy=individual_sites+individual_adp /
> anisotropic for all residues and isotropic for water /
>set_b_iso=10 / optimize_xyz/adp_weight=true
>   results: Rwork/free=0.2183/0.2706  b-
> factor=10.9/144.6/45.7 (min/max/average)
>   PS: the results is read from pdb file
> 
>   The results showed that the strategy of no5 is the
> best one. But the "B factor variance Graphs" generated by coot with
> menu/validate/temp.fact.variance analysis, have shown that no6 have the
> lowest B-factors (the attached figure is the B-factor graphs of three pdb
> files). And my questions are:
>   1. Which strategy should I choose to refine my
> structure? Or any other suggestions to refine the structure at 2.2A
> resolution?
>   2. Does it possible that some residues have very high
> 

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

I have seen anomalous "flecks" bespangling an protein's internal cavity which 
had a couple of cysteines in it, and I assumed that these were liberated 
sulphurs (they were not Fourier-truncation-like.) I would agree with Andrew 
that diffusion should not be large, but alighting on the nearest perch should 
be possible, no? Attractive and repulsive forces remain just as strong at 100K, 
so although motions are not driven by thermal sampling or random-walking, they 
would still be driven by local forces.

Further, I would think that the likeliest local perches would appear as new 
sites, for the CO2 as well as for sulphurs.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of DUMAS 
Philippe (VIE)
Sent: Thursday, May 04, 2017 6:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?

 
Le Jeudi 4 Mai 2017 12:25 CEST, Andrew Leslie  a 
écrit: 

Dear Andrew,
We looked in details to this problem of diffusion at ca. 100 K with bromine in 
"Ennifar et al., Acta D58(2002)1262" and we concluded

"It was attempted to derive a value for the diffusion coefficient of the free 
bromine species (most likely Br-) in amorphous ice at 100±110 K. This failed 
because the diffusion was much too rapid compared with both the radiolysis and 
datacollection timescales to permit such a determination."

Best regards
Philippe Dumas
 
> Dear Ed,
> 
>   I find your electron density quite interesting, because 
> generally (I think, I would be happy to be corrected on this) when 
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
> evidence of what happens to the resulting CO2 group. One interpretation of 
> this is that it diffuses away from the side chain and is effectively totally 
> disordered, so no electron density is seen, but I was surprised that this 
> would always be the case, especially as I would have thought that diffusion 
> would be quite limited at 100K (maybe I’m wrong about that too, but that is 
> supposed to be one reason why radiation damage is less at 100K).
> 
> If the residual density is due to partial de-carboxylation, then I would have 
> expected density for the CG-CD bond, which is not present (at your chosen 
> contour level).
> 
> Do many of your Glu side chains have the residual density?
> 
> Best wishes,
> 
> Andrew
> 
> 
> > On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> > 
> > 
> > 
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >> 
> >>  Have you considered the possibility that it could be a water

> >> stepping in to fill the void created by partial decarboxylation of 
> >> the glutamate? That could be easily modelled, refined, and tested 
> >> for its ability to flatten the difference map.
> >> 
> >>  Gerard.
> >> 
> > Actually some of them do appear decarboxylated. Is that something that can 
> > happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears 
> > continuous and linear, doesn't break up into spheres at H-bonding distance 
> > - almost like the CO2 is still sitting there- but I guess it would get 
> > hydrated to bicarbonate. I could use azide. Or maybe waters with some 
> > disorder.
> > Thanks,
> > eab
> > 
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> > comparison, not part of the model.
> > 
> > 
 
 
 
 

Re: [ccp4bb] very high B-factor

[Vipul Panchal]

1) the difference is 5.23 % and it is good and acceptable.
2) if higher value belongs to residue, then you need to change the rotamer
Using coot ,  follow menu-->distance-->residue info. You will get to know
to whom 417 B factor value belongs. Meanwhile, just check if modeled
residue or part of residue has relevant density. If density is missing for
part of residue, then set occupancy to zero for the relevant atom(s). If
density is missing for the model of residue, then just remove side chain. I
would recommend to check density fit (coot-->menu--> validate--> density
fit) for all residues with >50 B factor value (coot-->menu--> validate-->
temp. fact. variance analysis).


On 04-May-2017 3:26 PM, "yanqiaoling2782048" 
wrote:

Hi Vipul,

Thanks for your quick reply.

1. Actually, i mainly comparing the gap between Rwork and Rfree which
should be <5%, and then the overall b-factor, RMSD of bond and angle. For
me, no5 is the best one, but the "B factor variance Graphs" make me
uncomfortable.
The R-factor gap of no6 is 6.23%, does this mean overfitting?

2. The b-factor value of 76 is belong to the atom of the residue obtained
from the pdb file. I'm not sure whether the B-factor in  "B factor variance
Graphs" belong to residue or atom, but i guess 417 belongs to residue.
What's the relationship of the two factor?

Best regards,
Qiaoling Yan


At 2017-05-04 15:58:36, "Vipul Panchal"  wrote:

Hi,

1>
Well, it seems you are just comparing Max value across all protocols. You
should compare average values as it also takes into account no. of atoms
with given values of B factor. As per refinement results, it seems though
no. 6 has highest B factor value, number of atomic outliers are relatively
low. So if you ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you
should go ahead with no.6 is strategy.

2>
When comparing B factor values of 417 vs 76, you should consider what it
belongs to. As i may guess, 417 value belongs to an atom of residue whereas
76 belongs to residue. Meaning 417 is individual B factor whereas 76 is
grouped B factor.


On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048 <
yanqiaoling2782...@126.com> wrote:

> Dear all,
>
> I'm working on a crystal structure with resolution of 2.2A. At the final
> step, I use different strategies to refine the structure, they are:
> no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
> results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)
>
> no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 /
> optimize_xyz/adp_weight=true
> results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/m
> ax/average)
>
> no6: strategy=individual_sites+individual_adp / anisotropic for all
> residues and isotropic for water /
>  set_b_iso=10 / optimize_xyz/adp_weight=true
> results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7
> (min/max/average)
> PS: the results is read from pdb file
>
> The results showed that the strategy of no5 is the best one. But the "B
> factor variance Graphs" generated by coot with
> menu/validate/temp.fact.variance analysis, have shown that no6 have the
> lowest B-factors (the attached figure is the B-factor graphs of three pdb
> files). And my questions are:
> 1. Which strategy should I choose to refine my structure? Or any other
> suggestions to refine the structure at 2.2A resolution?
> 2. Does it possible that some residues have very high B-factor in "B
> factor variance Graphs", while in the pdb file， the b-factor of
> corresponding residues are relatively low? For example, one residue have
> B-factors of 417 in "B factor variance Graphs", but in PDB file the b
> factor is 76. Does the two factor mean the same thing?
> 3. If i want to set the weight manually, which parameter should i set,
> wxc/wxc_scale? or others?
>
> Thanks in advance.
>
> Best regards,
> Qiaoling Yan
>
>
>
>
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] peroxy-glutamate?

[VIE]

Le Jeudi 4 Mai 2017 12:25 CEST, Andrew Leslie  a 
écrit:

Dear Andrew,
We looked in details to this problem of diffusion at ca. 100 K with bromine in 
"Ennifar et al., Acta D58(2002)1262" and we concluded

"It was attempted to derive a value for the diffusion coefficient of the free 
bromine species (most likely Br-) in amorphous ice at 100±110 K. This failed 
because the diffusion was much too rapid compared with both the radiolysis and 
datacollection timescales to permit such a determination."

Best regards
Philippe Dumas

> Dear Ed,
>
>   I find your electron density quite interesting, because 
> generally (I think, I would be happy to be corrected on this) when 
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
> evidence of what happens to the resulting CO2 group. One interpretation of 
> this is that it diffuses away from the side chain and is effectively totally 
> disordered, so no electron density is seen, but I was surprised that this 
> would always be the case, especially as I would have thought that diffusion 
> would be quite limited at 100K (maybe I’m wrong about that too, but that is 
> supposed to be one reason why radiation damage is less at 100K).
>
> If the residual density is due to partial de-carboxylation, then I would have 
> expected density for the CG-CD bond, which is not present (at your chosen 
> contour level).
>
> Do many of your Glu side chains have the residual density?
>
> Best wishes,
>
> Andrew
>
>
> > On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> >
> >
> >
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >>
> >>  Have you considered the possibility that it could be a water

> >> stepping in to fill the void created by partial decarboxylation of the
> >> glutamate? That could be easily modelled, refined, and tested for its
> >> ability to flatten the difference map.
> >>
> >>  Gerard.
> >>
> > Actually some of them do appear decarboxylated. Is that something that can 
> > happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears 
> > continuous and linear, doesn't break up into spheres at H-bonding distance 
> > - almost like the CO2 is still sitting there- but I guess it would get 
> > hydrated to bicarbonate. I could use azide. Or maybe waters with some 
> > disorder.
> > Thanks,
> > eab
> >
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> > comparison, not part of the model.
> >
> > 

Re: [ccp4bb] peroxy-glutamate?

[Andrew Leslie]

Dear Ed,

  I find your electron density quite interesting, because generally 
(I think, I would be happy to be corrected on this) when de-carboxylation of 
Asp/Glu occurs due to radiation damage, there is no evidence of what happens to 
the resulting CO2 group. One interpretation of this is that it diffuses away 
from the side chain and is effectively totally disordered, so no electron 
density is seen, but I was surprised that this would always be the case, 
especially as I would have thought that diffusion would be quite limited at 
100K (maybe I’m wrong about that too, but that is supposed to be one reason why 
radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew


> On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> 
> 
> 
> On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
>> Dear Ed,
>> 
>>  Have you considered the possibility that it could be a water
>> stepping in to fill the void created by partial decarboxylation of the
>> glutamate? That could be easily modelled, refined, and tested for its
>> ability to flatten the difference map.
>> 
>>  Gerard.
>> 
> Actually some of them do appear decarboxylated. Is that something that can 
> happen? In the crystal, or as radiation damage?
> However when there is density for the carboxylate (figure), it appears 
> continuous and linear, doesn't break up into spheres at H-bonding distance - 
> almost like the CO2 is still sitting there- but I guess it would get hydrated 
> to bicarbonate. I could use azide. Or maybe waters with some disorder.
> Thanks,
> eab
> 
> Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> comparison, not part of the model.
> 
> 

Re: [ccp4bb] very high B-factor

[yanqiaoling2782048]

Hi Vipul,


Thanks for your quick reply.


1. Actually, i mainly comparing the gap between Rwork and Rfree which should be 
<5%, and then the overall b-factor, RMSD of bond and angle. For me, no5 is the 
best one, but the "B factor variance Graphs" make me uncomfortable. The 
R-factor gap of no6 is 6.23%, does this mean overfitting?


2. The b-factor value of 76 is belong to the atom of the residue obtained from 
the pdb file. I'm not sure whether the B-factor in  "B factor variance Graphs" 
belong to residue or atom, but i guess 417 belongs to residue. What's the 
relationship of the two factor?  


Best regards,
Qiaoling Yan 




At 2017-05-04 15:58:36, "Vipul Panchal"  wrote:

Hi,


1>
Well, it seems you are just comparing Max value across all protocols. You 
should compare average values as it also takes into account no. of atoms with 
given values of B factor. As per refinement results, it seems though no. 6 has 
highest B factor value, number of atomic outliers are relatively low. So if you 
ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you should 
go ahead with no.6 is strategy.


2>
When comparing B factor values of 417 vs 76, you should consider what it 
belongs to. As i may guess, 417 value belongs to an atom of residue whereas 76 
belongs to residue. Meaning 417 is individual B factor whereas 76 is grouped B 
factor.




On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048  
wrote:

Dear all,

I'm working on a crystal structure with resolution of 2.2A. At the final step, 
I use different strategies to refine the structure, they are:
no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)

no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 / 
optimize_xyz/adp_weight=true
results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/max/average)

no6: strategy=individual_sites+individual_adp / anisotropic for all residues 
and isotropic for water /  
 set_b_iso=10 / optimize_xyz/adp_weight=true
results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7 (min/max/average)
PS: the results is read from pdb file

The results showed that the strategy of no5 is the best one. But the "B factor 
variance Graphs" generated by coot with menu/validate/temp.fact.variance 
analysis, have shown that no6 have the lowest B-factors (the attached figure is 
the B-factor graphs of three pdb files). And my questions are:
1. Which strategy should I choose to refine my structure? Or any other 
suggestions to refine the structure at 2.2A resolution?
2. Does it possible that some residues have very high B-factor in "B factor 
variance Graphs", while in the pdb file， the b-factor of corresponding residues 
are relatively low? For example, one residue have B-factors of 417 in "B factor 
variance Graphs", but in PDB file the b factor is 76. Does the two factor mean 
the same thing?
3. If i want to set the weight manually, which parameter should i set, 
wxc/wxc_scale? or others?

Thanks in advance.

Best regards,
Qiaoling Yan






 






--

Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] very high B-factor

[Vipul Panchal]

Hi,

1>
Well, it seems you are just comparing Max value across all protocols. You
should compare average values as it also takes into account no. of atoms
with given values of B factor. As per refinement results, it seems though
no. 6 has highest B factor value, number of atomic outliers are relatively
low. So if you ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you
should go ahead with no.6 strategy.

2>
When comparing B factor values of 417 vs 76, you should consider what it
belongs to. As i may guess, 417 value belongs to an atom of residue whereas
76 belongs to residue. Meaning 417 is individual B factor whereas 76 is
grouped B factor.


On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048 <
yanqiaoling2782...@126.com> wrote:

> Dear all,
>
> I'm working on a crystal structure with resolution of 2.2A. At the final
> step, I use different strategies to refine the structure, they are:
> no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
> results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)
>
> no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 /
> optimize_xyz/adp_weight=true
> results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/
> max/average)
>
> no6: strategy=individual_sites+individual_adp / anisotropic for all
> residues and isotropic for water /
>  set_b_iso=10 / optimize_xyz/adp_weight=true
> results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7
> (min/max/average)
> PS: the results is read from pdb file
>
> The results showed that the strategy of no5 is the best one. But the "B
> factor variance Graphs" generated by coot with 
> menu/validate/temp.fact.variance
> analysis, have shown that no6 have the lowest B-factors (the attached
> figure is the B-factor graphs of three pdb files). And my questions are:
> 1. Which strategy should I choose to refine my structure? Or any other
> suggestions to refine the structure at 2.2A resolution?
> 2. Does it possible that some residues have very high B-factor in "B
> factor variance Graphs", while in the pdb file， the b-factor of
> corresponding residues are relatively low? For example, one residue have
> B-factors of 417 in "B factor variance Graphs", but in PDB file the b
> factor is 76. Does the two factor mean the same thing?
> 3. If i want to set the weight manually, which parameter should i set,
> wxc/wxc_scale? or others?
>
> Thanks in advance.
>
> Best regards,
> Qiaoling Yan
>
>
>
>
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372