Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

Try MR with one copy in all space groups of PG 321/312 using Phaser. Going from 
PG 3 to PG 32 should halve the number of copies per ASU. You may have to 
re-process your data in the higher point group to do this.

Or you might actually have a tetartohedral twin, but just try with the 
higher-symmetry point group first, see what happens.

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com]
Sent: Thursday, April 13, 2017 11:32 PM
To: Keller, Jacob 
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

Thanks for the suggestions! I only have two copies in ASU at SG P32. Zanuda 
also suggests P32 is the best SG.

On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in these types of things!

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK

Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in log 
file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 



Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
-L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
-L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
L;  H+K, -H,  L

On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst. (2017). 
D73, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Eleanor Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
mailto:r.joos...@nki.nl>> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solve

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Alex Lee]

Hi Keller,

Thanks for the suggestions! I only have two copies in ASU at SG P32. Zanuda
also suggests P32 is the best SG.

On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob 
wrote:

> Yes, this was my case exactly—it looks like there are two pairs of coupled
> twin domains: a,c and b,d. Assuming you have multiple copies of your model
> in the same ASU, try doing MR in higher symmetry space groups of point
> group 312 or 321, like P3212 etc. There is this handy page with all the
> space groups and their possible twin operators:
> http://www.ccp4.ac.uk/html/twinning.html.
>
>
>
> The twin fractions indicate a high twin fraction—~46% if actually
> hemihedral!
>
>
>
> Also take a look at the paper I referenced for more info. I can send you a
> .pdf if you need me to.
>
>
>
> Please let me know how it works out—I am interested in these types of
> things!
>
>
>
> JPK
>
>
>
> *From:* Alex Lee [mailto:alexlee198...@gmail.com]
> *Sent:* Thursday, April 13, 2017 9:08 PM
> *To:* Keller, Jacob 
> *Cc:* CCP4BB@JISCMAIL.AC.UK
>
> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree
> surprisingly down
>
>
>
> Hi Keller,
>
>
>
> I do not how to check twin fraction after Refmac (I guess it's somewhere
> in log file). From the log file it seems I have four twin domain:
>
>Twin operators with estimated twin fractions 
>
>
>
> Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
> L; -H-K,  H,  L
>
> Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
> -L;  H+K, -K, -L
>
> Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
> -L; -H-K,  K, -L
>
> Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
> L;  H+K, -H,  L
>
>
>
> On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
> wrote:
>
> What was the refined twin fraction after Refmac? It’s much more accurate
> than initial tests. Also, how many twin domains do you have? If you have
> many, it might be a higher space group but with less twinning. I recently
> had a case in which apparent tetartohedral (four-domain) twinning in P32
> was really hemihedral (two-domain) twinning in P3212:
>
>
>
> *Acta Cryst. * (2017). D*73*
> , 22-31
> https://doi.org/10.1107/S2059798316019318
>
>
>
> Jacob
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Eleanor
> Dodson
> *Sent:* Thursday, April 13, 2017 3:11 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree
> surprisingly down
>
>
>
> Twin refinement cannot be compared directly to untwinned - the R factors
> are between different parameters - without twinning it is assumed you have
> an amplitude obtained more or less from sqrt(I   But for a twinned data set
> that I is actually [ I1 + twin_factor I2 ] so the amplitude is not really
> correct and twinned refinement will give a much better estimate.
>
>
>
> However you need to be careful that you have assigned the same FreeR flag
> to reflection pair related by the twin law. The modern program in the CCP4
> data reduction pipeline looks after this pretty automatically - all
> possible symmetry equivalents are assigned the same FreeR but older
> software did not do this..
>
>
>
> You can check it by looking at some twin equivalents - in SG P32 these
> could be h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .
>
>
>
> Ideally they all should have the same Free R flag..
>
>
>
> Eleanor
>
>
>
> PS - the acid test is:  Do the maps look better?
>
>
>
> E
>
>
>
>
>
> On 13 April 2017 at 19:52, Robbie Joosten  wrote:
>
> Hi Alex,
>
>
>
> You are not giving the number after  refinement without the twin
> refinement. Nevertheless, R-free drops like this are not unheard of. You
> should check your Refmac log file, it would warn you of potential space
> group errors. Refmac will also give you a refined estimate of the twin
> fraction.
>
>
>
> Cheers,
>
> Robbie
>
>
>
> Sent from my Windows 10 phone
>
>
>
> *Van: *Alex Lee 
> *Verzonden: *donderdag 13 april 2017 19:19
> *Aan: *CCP4BB@JISCMAIL.AC.UK
> *Onderwerp: *[ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly
> down
>
>
>
> Dear All,
>
>
>
> I have a protein/dna complex crystal and data collected at 3A and another
> set at 2.8A, space group P32. L test shows twinning (fraction around 0.11).
> The structure solved by MR and model building of the complex finish (no
> solvent built yet, I do not think it's good to build solvent in such low
> resolution data).
>
>
>
> I did Refmac5 to refine my structure (restraint refinement) with or
> without twinning, to my surprise, the Rfree drops a lot after twin
> refinement of two data sets.  Summary below:
>
>
>
> 2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
>
> 3A dataset: before twin refine 30%;26%; after refine 25%, 18%
>
>
>
> I know that a lot of threads in CCP4bb talking about Rfree

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

Yes, this was my case exactly—it looks like there are two pairs of coupled twin 
domains: a,c and b,d. Assuming you have multiple copies of your model in the 
same ASU, try doing MR in higher symmetry space groups of point group 312 or 
321, like P3212 etc. There is this handy page with all the space groups and 
their possible twin operators: http://www.ccp4.ac.uk/html/twinning.html.

The twin fractions indicate a high twin fraction—~46% if actually hemihedral!

Also take a look at the paper I referenced for more info. I can send you a .pdf 
if you need me to.

Please let me know how it works out—I am interested in these types of things!

JPK

From: Alex Lee [mailto:alexlee198...@gmail.com]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob 
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in log 
file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 



Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K, -H-K,  
L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,  H+K, 
-L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H, -H-K, 
-L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,  H+K,  
L;  H+K, -H,  L

On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:
What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst. (2017). 
D73, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Eleanor Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
mailto:r.joos...@nki.nl>> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solved by MR and model building of the complex finish (no solvent 
built yet, I do not think it's good to build solvent in such low resolution 
data).

I did Refmac5 to refine my structure (restraint refinement) with or without 
twinning, to my surprise, the Rfree drops a lot after twin refinement of two 
data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin refine 
and Rfree without twin refine can not compare directly. By drop R free this 
much by twin refine, it gives me a feeling of too good to be true (at such low 
resolution with such good Rfree, maybe overrefined a lot?), but from the 
density map after twin refine, it does seem better than no twin refine map.

I do not know if r

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Alex Lee]

Hi Keller,

I do not how to check twin fraction after Refmac (I guess it's somewhere in
log file). From the log file it seems I have four twin domain:

   Twin operators with estimated twin fractions 


Twin operator:  H,  K,  L: Fraction = 0.275; Equivalent operators:  K,
-H-K,  L; -H-K,  H,  L

Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H,
 H+K, -L;  H+K, -K, -L

Twin operator:  K,  H, -L: Fraction = 0.270; Equivalent operators:  H,
-H-K, -L; -H-K,  K, -L

Twin operator: -H, -K,  L: Fraction = 0.228; Equivalent operators: -K,
 H+K,  L;  H+K, -H,  L


On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob 
wrote:

> What was the refined twin fraction after Refmac? It’s much more accurate
> than initial tests. Also, how many twin domains do you have? If you have
> many, it might be a higher space group but with less twinning. I recently
> had a case in which apparent tetartohedral (four-domain) twinning in P32
> was really hemihedral (two-domain) twinning in P3212:
>
>
>
> *Acta Cryst. * (2017). D*73*
> , 22-31
> https://doi.org/10.1107/S2059798316019318
>
>
>
> Jacob
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Eleanor
> Dodson
> *Sent:* Thursday, April 13, 2017 3:11 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree
> surprisingly down
>
>
>
> Twin refinement cannot be compared directly to untwinned - the R factors
> are between different parameters - without twinning it is assumed you have
> an amplitude obtained more or less from sqrt(I   But for a twinned data set
> that I is actually [ I1 + twin_factor I2 ] so the amplitude is not really
> correct and twinned refinement will give a much better estimate.
>
>
>
> However you need to be careful that you have assigned the same FreeR flag
> to reflection pair related by the twin law. The modern program in the CCP4
> data reduction pipeline looks after this pretty automatically - all
> possible symmetry equivalents are assigned the same FreeR but older
> software did not do this..
>
>
>
> You can check it by looking at some twin equivalents - in SG P32 these
> could be h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .
>
>
>
> Ideally they all should have the same Free R flag..
>
>
>
> Eleanor
>
>
>
> PS - the acid test is:  Do the maps look better?
>
>
>
> E
>
>
>
>
>
> On 13 April 2017 at 19:52, Robbie Joosten  wrote:
>
> Hi Alex,
>
>
>
> You are not giving the number after  refinement without the twin
> refinement. Nevertheless, R-free drops like this are not unheard of. You
> should check your Refmac log file, it would warn you of potential space
> group errors. Refmac will also give you a refined estimate of the twin
> fraction.
>
>
>
> Cheers,
>
> Robbie
>
>
>
> Sent from my Windows 10 phone
>
>
>
> *Van: *Alex Lee 
> *Verzonden: *donderdag 13 april 2017 19:19
> *Aan: *CCP4BB@JISCMAIL.AC.UK
> *Onderwerp: *[ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly
> down
>
>
>
> Dear All,
>
>
>
> I have a protein/dna complex crystal and data collected at 3A and another
> set at 2.8A, space group P32. L test shows twinning (fraction around 0.11).
> The structure solved by MR and model building of the complex finish (no
> solvent built yet, I do not think it's good to build solvent in such low
> resolution data).
>
>
>
> I did Refmac5 to refine my structure (restraint refinement) with or
> without twinning, to my surprise, the Rfree drops a lot after twin
> refinement of two data sets.  Summary below:
>
>
>
> 2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
>
> 3A dataset: before twin refine 30%;26%; after refine 25%, 18%
>
>
>
> I know that a lot of threads in CCP4bb talking about Rfree after twin
> refine and Rfree without twin refine can not compare directly. By drop R
> free this much by twin refine, it gives me a feeling of too good to be true
> (at such low resolution with such good Rfree, maybe overrefined a lot?),
> but from the density map after twin refine, it does seem better than no
> twin refine map.
>
>
>
> I do not know if reviewers are going to challenge this part.
>
>
>
> Any input is appreciated.
>
>
>
>
>
>
>
>
>

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Keller, Jacob]

What was the refined twin fraction after Refmac? It’s much more accurate than 
initial tests. Also, how many twin domains do you have? If you have many, it 
might be a higher space group but with less twinning. I recently had a case in 
which apparent tetartohedral (four-domain) twinning in P32 was really 
hemihedral (two-domain) twinning in P3212:

Acta Cryst. (2017). 
D73, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor 
Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Twin refinement cannot be compared directly to untwinned - the R factors are 
between different parameters - without twinning it is assumed you have an 
amplitude obtained more or less from sqrt(I   But for a twinned data set that I 
is actually [ I1 + twin_factor I2 ] so the amplitude is not really correct and 
twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag to 
reflection pair related by the twin law. The modern program in the CCP4 data 
reduction pipeline looks after this pretty automatically - all possible 
symmetry equivalents are assigned the same FreeR but older software did not do 
this..

You can check it by looking at some twin equivalents - in SG P32 these could be 
h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten 
mailto:r.joos...@nki.nl>> wrote:
Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solved by MR and model building of the complex finish (no solvent 
built yet, I do not think it's good to build solvent in such low resolution 
data).

I did Refmac5 to refine my structure (restraint refinement) with or without 
twinning, to my surprise, the Rfree drops a lot after twin refinement of two 
data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin refine 
and Rfree without twin refine can not compare directly. By drop R free this 
much by twin refine, it gives me a feeling of too good to be true (at such low 
resolution with such good Rfree, maybe overrefined a lot?), but from the 
density map after twin refine, it does seem better than no twin refine map.

I do not know if reviewers are going to challenge this part.

Any input is appreciated.

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Eleanor Dodson]

Twin refinement cannot be compared directly to untwinned - the R factors
are between different parameters - without twinning it is assumed you have
an amplitude obtained more or less from sqrt(I   But for a twinned data set
that I is actually [ I1 + twin_factor I2 ] so the amplitude is not really
correct and twinned refinement will give a much better estimate.

However you need to be careful that you have assigned the same FreeR flag
to reflection pair related by the twin law. The modern program in the CCP4
data reduction pipeline looks after this pretty automatically - all
possible symmetry equivalents are assigned the same FreeR but older
software did not do this..

You can check it by looking at some twin equivalents - in SG P32 these
could be h k l and -h, -k, l or h k l and k h -l  or h k l and -k, -h, -l .

Ideally they all should have the same Free R flag..

Eleanor

PS - the acid test is:  Do the maps look better?

E


On 13 April 2017 at 19:52, Robbie Joosten  wrote:

> Hi Alex,
>
>
>
> You are not giving the number after  refinement without the twin
> refinement. Nevertheless, R-free drops like this are not unheard of. You
> should check your Refmac log file, it would warn you of potential space
> group errors. Refmac will also give you a refined estimate of the twin
> fraction.
>
>
>
> Cheers,
>
> Robbie
>
>
>
> Sent from my Windows 10 phone
>
>
>
> *Van: *Alex Lee 
> *Verzonden: *donderdag 13 april 2017 19:19
> *Aan: *CCP4BB@JISCMAIL.AC.UK
> *Onderwerp: *[ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly
> down
>
>
> Dear All,
>
> I have a protein/dna complex crystal and data collected at 3A and another
> set at 2.8A, space group P32. L test shows twinning (fraction around 0.11).
> The structure solved by MR and model building of the complex finish (no
> solvent built yet, I do not think it's good to build solvent in such low
> resolution data).
>
> I did Refmac5 to refine my structure (restraint refinement) with or
> without twinning, to my surprise, the Rfree drops a lot after twin
> refinement of two data sets.  Summary below:
>
> 2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
> 3A dataset: before twin refine 30%;26%; after refine 25%, 18%
>
> I know that a lot of threads in CCP4bb talking about Rfree after twin
> refine and Rfree without twin refine can not compare directly. By drop R
> free this much by twin refine, it gives me a feeling of too good to be true
> (at such low resolution with such good Rfree, maybe overrefined a lot?),
> but from the density map after twin refine, it does seem better than no
> twin refine map.
>
> I do not know if reviewers are going to challenge this part.
>
> Any input is appreciated.
>
>
>
>

Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Robbie Joosten]

Hi Alex,

You are not giving the number after  refinement without the twin refinement. 
Nevertheless, R-free drops like this are not unheard of. You should check your 
Refmac log file, it would warn you of potential space group errors. Refmac will 
also give you a refined estimate of the twin fraction.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Alex Lee
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another set 
at 2.8A, space group P32. L test shows twinning (fraction around 0.11). The 
structure solved by MR and model building of the complex finish (no solvent 
built yet, I do not think it's good to build solvent in such low resolution 
data).

I did Refmac5 to refine my structure (restraint refinement) with or without 
twinning, to my surprise, the Rfree drops a lot after twin refinement of two 
data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin refine 
and Rfree without twin refine can not compare directly. By drop R free this 
much by twin refine, it gives me a feeling of too good to be true (at such low 
resolution with such good Rfree, maybe overrefined a lot?), but from the 
density map after twin refine, it does seem better than no twin refine map.

I do not know if reviewers are going to challenge this part.

Any input is appreciated.

[ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

[Alex Lee]

Dear All,

I have a protein/dna complex crystal and data collected at 3A and another
set at 2.8A, space group P32. L test shows twinning (fraction around 0.11).
The structure solved by MR and model building of the complex finish (no
solvent built yet, I do not think it's good to build solvent in such low
resolution data).

I did Refmac5 to refine my structure (restraint refinement) with or without
twinning, to my surprise, the Rfree drops a lot after twin refinement of
two data sets.  Summary below:

2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19%
3A dataset: before twin refine 30%;26%; after refine 25%, 18%

I know that a lot of threads in CCP4bb talking about Rfree after twin
refine and Rfree without twin refine can not compare directly. By drop R
free this much by twin refine, it gives me a feeling of too good to be true
(at such low resolution with such good Rfree, maybe overrefined a lot?),
but from the density map after twin refine, it does seem better than no
twin refine map.

I do not know if reviewers are going to challenge this part.

Any input is appreciated.

[ccp4bb] 12 PhD places available in Membrane Protein studies!

[Goldman, Adrian]

There are 12 PhD places available as part of the EU Innovative Training Network 
“RAMP” (RAtionalising Membrane Protein Crystallisation), headed by Monika Spano.

Applicants can be from anywhere in the world, but can not have worked in the 
country where they want to get a PhD in the last three years.  The topics cover 
everything from mathematical modelling, developing novel devices, structural 
studies of transporters and receptors to use of the European Spallation Source, 
and the industrial partners include both Novartis and Astra-Zeneca.

To apply, and for more details of the positions, see 
www.ramp-itn.eu.  The current closing date for 
applications is April 23rd.

[ccp4bb] Deadline approaching: 10th CCP4/APS Crystallographic School in the USA

[Sanishvili, Ruslan]

Dear Colleagues,
The deadline for application to the annual CCP4/APS school in macromolecular 
crystallography: “From data collection to structure refinement and beyond” is 
in less than a week - April 19.
Please read the thread below for basic information on the school. For all 
details, please visit http://www.ccp4.ac.uk/schools/APS-2017/index.php
With regards,
Garib, Ronan and Nukri


Ruslan Sanishvili (Nukri), Ph.D.
Macromolecular Crystallographer
GM/CA@APS
X-ray Science Division, ANL
9700 S. Cass Ave.
Lemont, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, 
Ruslan [rsanishv...@anl.gov]
Sent: Monday, March 27, 2017 3:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] REMINDER: 10th CCP4/APS Crystallographic School in the USA

Dear Colleagues,

This is a friendly reminder for the 10th annual CCP4 crystallographic school 
“From data collection to structure refinement and beyond”, held from June 19 to 
26 at Advanced Photon Source (APS), Argonne National Laboratory (ANL). If you 
are interested attending, please take a look at the original announcement below 
for more details.
For all details, please visit the school web site 
http://www.ccp4.ac.uk/schools/APS-2017/index.php

With best wishes,
Garib, Ronan and Nukri

Ruslan Sanishvili (Nukri), Ph.D.
Macromolecular Crystallographer
GM/CA@APS
X-ray Science Division, ANL
9700 S. Cass Ave.
Lemont, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, 
Ruslan [rsanishv...@anl.gov]
Sent: Wednesday, February 01, 2017 2:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 10th CCP4/APS Crystallographic School in the USA

Dear Colleagues,

We are pleased to announce the 10th annual CCP4 crystallographic school “>From 
data collection to structure refinement and beyond”, held at Advanced Photon 
Source (APS), Argonne National Laboratory (ANL). All details can be found at 
http://www.ccp4.ac.uk/schools/APS-2017/index.php

Dates: June 19 through 26, 2017

Site: Advanced Photon Source, Argonne National Laboratory, Argonne (Near 
Chicago), Illinois, USA

The school comprises
Data collection workshop: beamline training; data collection on GM/CA@APS 
beamlines 23ID-D and 23ID-B equipped with Pilatus3 6M and Eiger 16M detectors, 
respectively; and data processing. For data collection, only the participants' 
crystals will be used.
Crystallographic computation workshop: will feature many modern 
crystallographic software packages taught by authors and other experts. The 
daily schedule will be organized in three sections – lectures, tutorials, and 
hands-on, interactive trouble-shooting of the technical difficulties the 
participants face in their projects. We have had considerable success resolving 
these problems in past years, attested by resulting publications 
http://www.ccp4.ac.uk/schools/APS-school/publications.php

Applicants: Graduate students, postdoctoral researchers and young scientists at 
the assistant professor level, along with commercial/industrial researchers are 
encouraged to apply. Only 20 applicants will be selected for participation. 
Participants of the workshop are strongly encouraged to bring their own problem 
data sets or crystals so the problems can be addressed during data collection 
and/or computation workshops.

Application: Application deadline is April 19. The application form, the 
program, contact info and other details can be found at 
http://www.ccp4.ac.uk/schools/APS-2017/index.php

Registration fees: The registration for application is free but there is $500 
participation fee for the selected academic students and $950 for the 
industrial researchers. The link for the on-line payments and instructions will 
be provided once the selection process is completed. The students will be 
responsible for their transportation, lodging and breakfast. The workshop 
organizers can assist in making the reservations at economical lodging at the 
Argonne Guest House. The workshop will cover all other expenses.

We hope to see you at the school.
Garib, Ronan and Nukri


Ruslan Sanishvili (Nukri), Ph.D.
Macromolecular Crystallographer
GM/CA@APS
X-ray Science Division, ANL
9700 S. Cass Ave.
Lemont, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov

Re: [ccp4bb] in crystallo enzymatic activity

[Bonsor, Daniel]

Are you using the same columns for purifying both WT and mutant? Could you have 
the tiniest fraction of WT contaminating your mutant batches which would then 
turnover you substrate in crystallo over the couple of weeks? You could try 
washing the columns/systems with NaOH or pepsin to remove WT, or just use 
separate columns. 

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pierre 
Nioche
Sent: Thursday, April 13, 2017 11:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] in crystallo enzymatic activity

Dear CCP4bb,

We work on an enzyme that we crystallized with two substrates bound in the 
active site (the reaction transform two substrates into two products). We have 
also the structure with the two products. We are able to see densities for the 
substrates when we collect data at different time point post-crystallization 
(days or weeks later). There is no change over time and no in crystallo 
enzymatic reaction despite the fact that in solution using the same 
crystallization solution, the reaction occurs readily.
This is not surprising and there are already many examples in the literature.
However, when we crystallize a single amino acid variant (mutant within the 
active site) with the same two substrates, we initially see the substrates but 
we then observe in crystallo enzymatic activity and formation of the final 
products over time. This structure is identical to the one determined with the 
two products co-crystallized with the enzyme. The crystal packing does not seem 
to be at play here.
I understand that in crystallo activities are well documented in the literature 
and can be induced by addition of ligands, X-rays, change in oxidative 
environment, etc?
Here, the substrates are present from the beginning of the crystallization 
experiments with the same concentration. Nothing is added to the crystals later 
on. Only the time differentiate the two type of crystals: after a couple of 
weeks, one has the substrates in the active site (wt) while the other has the 
products (variant).

Is anyone aware of similar examples where a variant induce in crystallo 
enzymatic activity without perturbation of the crystal?

Thanks,

Pierre
Dept of Pharmacology, Toxicology and cellular signaling Paris Descartes 
University

[ccp4bb] in crystallo enzymatic activity

[Pierre Nioche]

Dear CCP4bb,

We work on an enzyme that we crystallized with two substrates bound in  
the active site (the reaction transform two substrates into two  
products). We have also the structure with the two products. We are  
able to see densities for the substrates when we collect data at  
different time point post-crystallization (days or weeks later). There  
is no change over time and no in crystallo enzymatic reaction despite  
the fact that in solution using the same crystallization solution, the  
reaction occurs readily.

This is not surprising and there are already many examples in the literature.
However, when we crystallize a single amino acid variant (mutant  
within the active site) with the same two substrates, we initially see  
the substrates but we then observe in crystallo enzymatic activity and  
formation of the final products over time. This structure is identical  
to the one determined with the two products co-crystallized with the  
enzyme. The crystal packing does not seem to be at play here.
I understand that in crystallo activities are well documented in the  
literature and can be induced by addition of ligands, X-rays, change  
in oxidative environment, etc?
Here, the substrates are present from the beginning of the  
crystallization experiments with the same concentration. Nothing is  
added to the crystals later on. Only the time differentiate the two  
type of crystals: after a couple of weeks, one has the substrates in  
the active site (wt) while the other has the products (variant).


Is anyone aware of similar examples where a variant induce in  
crystallo enzymatic activity without perturbation of the crystal?


Thanks,

Pierre
Dept of Pharmacology, Toxicology and cellular signaling
Paris Descartes University

[ccp4bb] LAST CALL - 8th workshop on Neutron Scattering Applications in Structural Biology

[Meilleur, Flora]

LAST CALL

­­
8th Workshop on Neutron Scattering Applications in Structural Biology
Oak Ridge, TN. June 5 - June 9, 2017

Detailed information can be found on the workshop web page: 
https://conference.sns.gov/event/66/
Application deadline: April 22, 2017

The workshop on Neutron Scattering Applications in Structural Biology aims at 
enabling structural biologists to fully exploit the latest instrumentation and 
software development at the SNS and HFIR facilities at Oak Ridge National 
Laboratory. Attendees will participate in lectures and tutorials focusing 
exclusively on neutron techniques applied in structural biology. The workshop 
is designed for graduate students, post-doctoral fellows and faculty new to or 
with limited experience of neutron scattering.

Travel and accommodation expenses are supported for selected participants from 
the United States. International applications are welcome - however the course 
organization is not able to support travel or accommodation.

The number of participants will be limited to 15. There is no registration fee 
for all selected participants.

The application package consisting of 1) Information form, 2) CV, 3) Applicant 
motivation letter (1/2 to 1 page), 4) Principal Investigator letter of support 
(for graduate students only; 1/2 to 1 page), should be sent electronically to 
meille...@ornl.gov before April 22, 2017.

Flora Meilleur, Ph. D
Associate Professor, Molecular and Structural Biochemistry
North Carolina State University
Neutron Sciences Directorate
Oak Ridge National Laboratory

Re: [ccp4bb] waters with positive FoFc peaks?

[Robbie Joosten]

How are the peaks coordinated? Regular coordination with 5 or more ligands has 
ion written all over it. Different difference density peak heights can be 
caused by differences in effective B-factor restraint weight but also by 
differences in relative scattering factors of oxygen and whatever you have.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Keller, Jacob
Verzonden: donderdag 13 april 2017 13:05
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: Re: [ccp4bb] waters with positive FoFc peaks?

It would be useful to know what wavelengths you were talking about. Also, try 
an anomalous difference Fourier map to see whether the atoms are weakly 
anomalous.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew 
Marshall
Sent: Thursday, April 13, 2017 2:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] waters with positive FoFc peaks?

Hello all,

I have a 1.8A structure (Rfree/Rwork = 20.5/17.4) with 1420 water molecules 
modelled. There are approximately a dozen waters, all well structured with 
hydrogen bonds to protein atoms, with positive difference density (>4sigma, 
sometimes >5) at their centre. The only ions present in my buffer were Cs, Cl 
and a small amount of Na. I thought Cl ions might be a possibility, but many of 
them are in close proximity to acidic residues and/or one-another. It's 
probably worth noting that the same structure solved using data to 2.25A from 
the same crystal at a different wavelength doesn't contain these peaks (the 
offending waters look normal).

Has any come across this before? Thoughts?

Thanks,

Andrew Marshall
PhD Candidate
Laboratory of Protein Crystallography
Dept. of Molecular and Cellular Biology
School of Biological Sciences
The University of Adelaide

Re: [ccp4bb] waters with positive FoFc peaks?

[Keller, Jacob]

It would be useful to know what wavelengths you were talking about. Also, try 
an anomalous difference Fourier map to see whether the atoms are weakly 
anomalous.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew 
Marshall
Sent: Thursday, April 13, 2017 2:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] waters with positive FoFc peaks?

Hello all,

I have a 1.8A structure (Rfree/Rwork = 20.5/17.4) with 1420 water molecules 
modelled. There are approximately a dozen waters, all well structured with 
hydrogen bonds to protein atoms, with positive difference density (>4sigma, 
sometimes >5) at their centre. The only ions present in my buffer were Cs, Cl 
and a small amount of Na. I thought Cl ions might be a possibility, but many of 
them are in close proximity to acidic residues and/or one-another. It's 
probably worth noting that the same structure solved using data to 2.25A from 
the same crystal at a different wavelength doesn't contain these peaks (the 
offending waters look normal).

Has any come across this before? Thoughts?

Thanks,

Andrew Marshall
PhD Candidate
Laboratory of Protein Crystallography
Dept. of Molecular and Cellular Biology
School of Biological Sciences
The University of Adelaide

Re: [ccp4bb] waters with positive FoFc peaks?

[Mark J. van Raaij]

Partially occupied Cs+ comes to mind. At lower resolution the difference may be 
more easily fudged away by the refinement lowering the B.
Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij
 Original message From: Andrew Marshall 
 Date: 13/04/2017  07:59  (GMT+01:00) To: 
CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] waters with positive FoFc peaks? 
Hello all,
I have a 1.8A structure (Rfree/Rwork = 20.5/17.4) with 1420 water molecules 
modelled. There are approximately a dozen waters, all well structured with 
hydrogen bonds to protein atoms, with positive difference density (>4sigma, 
sometimes >5) at their centre. The only ions present in my buffer were Cs, Cl 
and a small amount of Na. I thought Cl ions might be a possibility, but many of 
them are in close proximity to acidic residues and/or one-another. It's 
probably worth noting that the same structure solved using data to 2.25A from 
the same crystal at a different wavelength doesn't contain these peaks (the 
offending waters look normal).
Has any come across this before? Thoughts?
Thanks,
Andrew MarshallPhD CandidateLaboratory of Protein CrystallographyDept. of 
Molecular and Cellular BiologySchool of Biological Sciences
The University of Adelaide