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

[Alex Lee]

Thanks Eleanor,
If I understand right, there is just 1 TFZ. TFZ== means "Translation
Function Z-score equivalent, only calculated for the top solution after
refinement (or for the number of top files specified by TOPFILES)" so there
could be many TFZ==.

Twinning analysis attached below:

TWINNING ANALYSIS:


Global twinning statistics.


These tests rely on the fact that it is highly improbably that very
weak or very strong reflections will coincide, therefore, the tails
for the distribution of twinned datasets will be less pronounced


Data truncated to  67.93 -   3.50 A resolution

$TABLE: Cumulative intensity distribution:

$GRAPHS: Cumulative intensity distribution (Acentric and centric):N:1,2,3,4,5,6:

$$ Z Acent_theor Acent_twin Acent_obser Cent_theor Cent_obser $$

$$

   0.0  0.0  0.0  0.01988  0.0   -

   0.04000  0.03921  0.00303  0.03225  0.15852   -

   0.08000  0.07688  0.01151  0.04745  0.22270   -

   0.12000  0.11308  0.02458  0.06655  0.27097   -

   0.16000  0.14786  0.04148  0.09008  0.31084   -

   0.2  0.18127  0.06155  0.11562  0.34528   -

   0.24000  0.21337  0.08420  0.14170  0.37579   -

   0.28000  0.24422  0.10891  0.16750  0.40330   -

   0.32000  0.27385  0.13524  0.19450  0.42839   -

   0.36000  0.30232  0.16279  0.22295  0.45149   -

   0.4  0.32968  0.19121  0.25213  0.47291   -

   0.44000  0.35596  0.22021  0.28103  0.49288   -

   0.48000  0.38122  0.24953  0.30678  0.51158   -

   0.52000  0.40548  0.27895  0.33697  0.52916   -

   0.56000  0.42879  0.30829  0.36569  0.54574   -

   0.6  0.45119  0.33737  0.39332  0.56142   -

   0.64000  0.47271  0.36607  0.41967  0.57629   -

   0.68000  0.49338  0.39428  0.44513  0.59041   -

   0.72000  0.51325  0.42190  0.47060  0.60386   -

   0.76000  0.53233  0.44885  0.49430  0.61667   -

   0.8  0.55067  0.47507  0.51550  0.62891   -

   0.84000  0.56829  0.50052  0.53736  0.64060   -

   0.88000  0.58522  0.52516  0.55780  0.65180   -

   0.92000  0.60148  0.54896  0.57822  0.66253   -

   0.96000  0.61711  0.57191  0.59779  0.67281   -

   1.0  0.63212  0.59399  0.61638  0.68269   -

   1.04000  0.64655  0.61521  0.63496  0.69218   -

   1.08000  0.66040  0.63557  0.65120  0.70130   -

   1.12000  0.67372  0.65507  0.66731  0.71008   -

   1.16000  0.68651  0.67373  0.68115  0.71853   -

   1.2  0.69881  0.69156  0.69557  0.72668   -

   1.24000  0.71062  0.70857  0.70990  0.73453   -

   1.28000  0.72196  0.72480  0.72386  0.74210   -

   1.32000  0.73286  0.74025  0.73801  0.74941   -

   1.36000  0.74334  0.75495  0.74895  0.75646   -

   1.4  0.75340  0.76892  0.75913  0.76328   -

   1.44000  0.76307  0.78220  0.77024  0.76986   -

   1.48000  0.77236  0.79480  0.78058  0.77623   -

   1.52000  0.78129  0.80675  0.79061  0.78238   -

   1.56000  0.78986  0.81807  0.79977  0.78833   -

   1.6  0.79810  0.82880  0.80902  0.79410   -

   1.64000  0.80602  0.83895  0.81795  0.79967   -

   1.68000  0.81363  0.84855  0.82691  0.80508   -

   1.72000  0.82093  0.85763  0.83511  0.81031   -

   1.76000  0.82796  0.86621  0.84245  0.81538   -

   1.8  0.83470  0.87431  0.85141  0.82029   -

   1.84000  0.84118  0.88196  0.85966  0.82505   -

   1.88000  0.84741  0.88917  0.86721  0.82967   -

   1.92000  0.85339  0.89597  0.87415  0.83414   -

   1.96000  0.85914  0.90238  0.87977  0.83849   -

   2.0  0.86466  0.90842  0.88629  0.84270   -

$$



The culmulative intensity, N(Z), plot is diagnostic for both twinning
and tNCS.  For twinned data there are fewer weak reflections,
therefore, N(Z) is sigmoidal for twinned data.  However, if both
twinning and tNCS are present, the effects may cancel each out.
Therefore the results of the L-test and patterson test should be
consulted



L test for twinning: (Padilla and Yeates Acta Cryst. D59 1124 (2003))

L statistic =  0.416  (untwinned 0.5 perfect twin 0.375)

Data has used to  67.93 -   3.50 A resolution

   Relation between L statistics and twinning fraction:

  Twinning fraction = 0.000  L statistics = 0.500:

  Twinning fraction = 0.100  L statistics = 0.440:

  Twinning fraction = 0.500  L statistics = 0.375:


The L test suggests data is twinned

All data regardless of I/sigma(I) has been included in the L test



$TABLE: L test for twinning:

$GRAPHS: cumulative distribution function for |L|, twin fraction of
0.18:0|1x0|1:1,2,3,4:

$$ |L|   N(L) Untwinned Twinned $$

$$

0. 0.  0.   0.

0.0500 0.0666  0.0500   0.0749

0.1000 0.1339  0.1000   0.1495

0.1500 0.2007  0.1500   0.2233

0.2000 0.2648  0.2000   0.2960

0.2500 0.3263  0.2500   0.3672

0.3000 0.3870  0.3000   0.4365

0.3500 0.4475  0.3500   0.5036

0.4000 0.5070  0.4000   0.5680

0.4500 0.5646  0.4500   0.6294

0.5000 0.6206  

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Peter Hsu]

Hi Sutapa,

With regards to insect cell expression, how do you know it wasn't expressed? 
You didn't see a band by coomassie staining or by western? 

If by staining, I'd recommend to just try expressing a limited amount through 
infection of a few 150mm dishes of your favorite cell line (I use High fives), 
and then do a quick lysis and purification over a small (200uL) column. In my 
experience, most of my proteins (including some big ones around 100kda) don't 
show up readily on a whole cell lysate, but you can actually see it and have 
quite a bit of it after seeing it on a column. While it's a pain, I find it far 
easier to get larger yields of protein from monolayer plates of insect cells vs 
suspension culture. Something about plates produces far happier cells = more 
protein. 

Are you using a His or GST tag for insect cells? My experience has always been 
use GST for insect cells, the purification is far simpler and your protein is 
much, much cleaner as a result. 

Hope it helps,
P

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

[Keller, Jacob]

It’s p3221. Re-process your data forcing this space group, rebuild, and refine 
twin domains. Just do it—you won’t regret it!

Jacob

From: Eleanor Dodson [mailto:eleanor.dod...@york.ac.uk]
Sent: Friday, April 14, 2017 3:19 PM
To: Keller, Jacob 
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

maybe attach your data processing log file..
e

On 14 April 2017 at 20:10, Eleanor Dodson 
> wrote:
That twin factor list  means the apparent crystal symmetry must be P6/mmm.

You say you only have 2 molecules in the asymmetric unit of P32,therefor there 
must only be one in SGs P32 21 P32 12

So I dont understand why you have PHASER results like this:

SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 LLG=350 
TFZ==20.5..



Why so many TFZ here - is that achieved after refinement or something?



Eleanor



And what does the twinning analysis suggest?





On 14 April 2017 at 17:42, Keller, Jacob 
> wrote:
As I mentioned off-list, it would be helpful to know how many types of search 
models you are searching with—how many different molecules are in the complex? 
It’s hard to interpret MR results otherwise.

Also, since the higher-symmetry SG works in MR, you should try to refine the 
model in that SG, with only two twin domains, refining twin fraction. I can 
guarantee that a good reviewer will have you do this (if not, then not a “good 
reviewer.”)

JPK

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex 
Lee
Sent: Friday, April 14, 2017 11:50 AM

To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Thanks Eleanor, I tried MR for P32 21 and P32 12.
SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944 TFZ==29.2 
PAK=0 LLG=944 TFZ==29.2

   SOLU SPAC P 32 2 1



SG P3212:

Solution #1 annotation (history):



   SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
LLG=350 TFZ==20.5



   SOLU SPAC P 32 1 2



SG P32

SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0 LLG=436 
TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0

LLG=1715 TFZ==34.3

   SOLU SPAC P 32



Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine and 
build P32 2 1 latter

On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
> wrote:
First - four way twinning is possible but pretty rare for macromolecules

Pointless gives a very useful table of the CC agreement for each possible 
symmetry operator individually.
In this case with only two molecules in the asymmetric unit you you could only 
have a higher symmetry SG as
P32 21 P32 12 or P64

These would require as symmetry operators -
P32 21 - a three fold and a two fold k h -l
P32 12 - a three fold and a two fold -k -h -l

P64 - a six fold

If the scores for one set are better than the others you probably have that SG

However high degrees of twinning can disguise the symmetry scores of course..



On 14 April 2017 at 04:46, Keller, Jacob 
> wrote:
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 
> 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 

Re: [ccp4bb] in crystallo enzymatic activity

[Petri Kursula]

Hi,

apart from the possibilities of conformational flexibility affected by crystal 
packing, just wondering if this mutation is supposed to actually cause an 
increase or decrease in the corresponding activity (outside the context of a 
crystal)? k(cat) and K(M) measured in solution would help here. Is the pH in 
the crystal far from the optimum of the wild-type protein? Can optimal pH 
change with the mutation?

Petri

Petri Kursula
--
Professor 
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula 

petri.kurs...@uib.no 
--
Group Leader, Adjunct Professor
Faculty of Biochemistry and Molecular Medicine
University of Oulu, Finland
--



> On 13 Apr 2017, at 17:27, Pierre Nioche  
> wrote:
> 
> 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

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

[Eleanor Dodson]

maybe attach your data processing log file..
e

On 14 April 2017 at 20:10, Eleanor Dodson  wrote:

> That twin factor list  means the apparent crystal symmetry must be P6/mmm.
>
> You say you only have 2 molecules in the asymmetric unit of P32,therefor
> there must only be one in SGs P32 21 P32 12
>
> So I dont understand why you have PHASER results like this:
>
> SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
> LLG=350 TFZ==20.5..
>
>
> Why so many TFZ here - is that achieved after refinement or something?
>
>
> Eleanor
>
>
> And what does the twinning analysis suggest?
>
>
>
>
> On 14 April 2017 at 17:42, Keller, Jacob  wrote:
>
>> As I mentioned off-list, it would be helpful to know how many types of
>> search models you are searching with—how many different molecules are in
>> the complex? It’s hard to interpret MR results otherwise.
>>
>>
>>
>> Also, since the higher-symmetry SG works in MR, you should try to refine
>> the model in that SG, with only two twin domains, refining twin fraction. I
>> can guarantee that a good reviewer will have you do this (if not, then not
>> a “good reviewer.”)
>>
>>
>>
>> JPK
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Alex Lee
>> *Sent:* Friday, April 14, 2017 11:50 AM
>>
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree
>> surprisingly down
>>
>>
>>
>> Thanks Eleanor, I tried MR for P32 21 and P32 12.
>>
>> SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944
>> TFZ==29.2 PAK=0 LLG=944 TFZ==29.2
>>
>>SOLU SPAC P 32 2 1
>>
>>
>>
>> SG P3212:
>>
>> Solution #1 annotation (history):
>>
>>
>>
>>SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
>> LLG=350 TFZ==20.5
>>
>>
>>
>>SOLU SPAC P 32 1 2
>>
>>
>>
>> SG P32
>>
>> SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0
>> LLG=436 TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0
>>
>> LLG=1715 TFZ==34.3
>>
>>SOLU SPAC P 32
>>
>>
>>
>> Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine 
>> and build P32 2 1 latter
>>
>>
>>
>> On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson <
>> eleanor.dod...@york.ac.uk> wrote:
>>
>> First - four way twinning is possible but pretty rare for macromolecules
>>
>>
>>
>> Pointless gives a very useful table of the CC agreement for each possible
>> symmetry operator individually.
>>
>> In this case with only two molecules in the asymmetric unit you you could
>> only have a higher symmetry SG as
>>
>> P32 21 P32 12 or P64
>>
>>
>>
>> These would require as symmetry operators -
>>
>> P32 21 - a three fold and a two fold k h -l
>>
>> P32 12 - a three fold and a two fold -k -h -l
>>
>>
>>
>> P64 - a six fold
>>
>>
>>
>> If the scores for one set are better than the others you probably have
>> that SG
>>
>>
>>
>> However high degrees of twinning can disguise the symmetry scores of
>> course..
>>
>>
>>
>>
>>
>>
>>
>> On 14 April 2017 at 04:46, Keller, Jacob 
>> wrote:
>>
>> 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 
>> 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
>> 

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

[Eleanor Dodson]

That twin factor list  means the apparent crystal symmetry must be P6/mmm.

You say you only have 2 molecules in the asymmetric unit of P32,therefor
there must only be one in SGs P32 21 P32 12

So I dont understand why you have PHASER results like this:

SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5
PAK=0 LLG=350 TFZ==20.5..


Why so many TFZ here - is that achieved after refinement or something?


Eleanor


And what does the twinning analysis suggest?




On 14 April 2017 at 17:42, Keller, Jacob  wrote:

> As I mentioned off-list, it would be helpful to know how many types of
> search models you are searching with—how many different molecules are in
> the complex? It’s hard to interpret MR results otherwise.
>
>
>
> Also, since the higher-symmetry SG works in MR, you should try to refine
> the model in that SG, with only two twin domains, refining twin fraction. I
> can guarantee that a good reviewer will have you do this (if not, then not
> a “good reviewer.”)
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Alex
> Lee
> *Sent:* Friday, April 14, 2017 11:50 AM
>
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree
> surprisingly down
>
>
>
> Thanks Eleanor, I tried MR for P32 21 and P32 12.
>
> SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944
> TFZ==29.2 PAK=0 LLG=944 TFZ==29.2
>
>SOLU SPAC P 32 2 1
>
>
>
> SG P3212:
>
> Solution #1 annotation (history):
>
>
>
>SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
> LLG=350 TFZ==20.5
>
>
>
>SOLU SPAC P 32 1 2
>
>
>
> SG P32
>
> SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0
> LLG=436 TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0
>
> LLG=1715 TFZ==34.3
>
>SOLU SPAC P 32
>
>
>
> Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine 
> and build P32 2 1 latter
>
>
>
> On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
> wrote:
>
> First - four way twinning is possible but pretty rare for macromolecules
>
>
>
> Pointless gives a very useful table of the CC agreement for each possible
> symmetry operator individually.
>
> In this case with only two molecules in the asymmetric unit you you could
> only have a higher symmetry SG as
>
> P32 21 P32 12 or P64
>
>
>
> These would require as symmetry operators -
>
> P32 21 - a three fold and a two fold k h -l
>
> P32 12 - a three fold and a two fold -k -h -l
>
>
>
> P64 - a six fold
>
>
>
> If the scores for one set are better than the others you probably have
> that SG
>
>
>
> However high degrees of twinning can disguise the symmetry scores of
> course..
>
>
>
>
>
>
>
> On 14 April 2017 at 04:46, Keller, Jacob  wrote:
>
> 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 
> 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 

[ccp4bb] Post doctoral positions at Pfizer, Groton CT - Protein Crystallography/Structural Biology

[Pandit, Jayvardhan]

Applications are invited for post-doctoral positions in my group at Pfizer's 
Research Laboratories in Groton CT. Please see the advertisements posted on 
LinkedIn:

https://www.linkedin.com/jobs/view/295904458/  and

https://www.linkedin.com/jobs/view/290461069/

Positions are available for highly motivated, outstanding applicants with a 
recent Ph.D and experience in protein crystallography, biochemistry and 
molecular biology, and an interest in working in a drug discovery group. You 
will work as part of a highly interdisciplinary structural biology and 
biophysics group with expertise in protein crystallography, protein NMR, 
cryo-electron microscopy, advanced biophysical and biochemical techniques and 
molecular biology, all working towards the goal of discovering new human 
therapeutics.

Groton  is located on the Connecticut shoreline, almost exactly midway between 
New York and Boston.

Please send your resume through the links to the company website provided in 
the advertisements.

--

Jay Pandit, Ph.D.
Research Fellow
Discovery Sciences, Worldwide Research and Development
Pfizer Inc
Eastern Point Road
Groton, CT 06340
USA
+1 860 441 3738

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

[Keller, Jacob]

As I mentioned off-list, it would be helpful to know how many types of search 
models you are searching with—how many different molecules are in the complex? 
It’s hard to interpret MR results otherwise.

Also, since the higher-symmetry SG works in MR, you should try to refine the 
model in that SG, with only two twin domains, refining twin fraction. I can 
guarantee that a good reviewer will have you do this (if not, then not a “good 
reviewer.”)

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex Lee
Sent: Friday, April 14, 2017 11:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly down

Thanks Eleanor, I tried MR for P32 21 and P32 12.
SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944 TFZ==29.2 
PAK=0 LLG=944 TFZ==29.2

   SOLU SPAC P 32 2 1



SG P3212:

Solution #1 annotation (history):



   SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 
LLG=350 TFZ==20.5



   SOLU SPAC P 32 1 2



SG P32

SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0 LLG=436 
TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0

LLG=1715 TFZ==34.3

   SOLU SPAC P 32



Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine and 
build P32 2 1 latter

On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
> wrote:
First - four way twinning is possible but pretty rare for macromolecules

Pointless gives a very useful table of the CC agreement for each possible 
symmetry operator individually.
In this case with only two molecules in the asymmetric unit you you could only 
have a higher symmetry SG as
P32 21 P32 12 or P64

These would require as symmetry operators -
P32 21 - a three fold and a two fold k h -l
P32 12 - a three fold and a two fold -k -h -l

P64 - a six fold

If the scores for one set are better than the others you probably have that SG

However high degrees of twinning can disguise the symmetry scores of course..



On 14 April 2017 at 04:46, Keller, Jacob 
> wrote:
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 
> 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 

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

[Alex Lee]

Thanks Eleanor, I tried MR for P32 21 and P32 12.
SG P3221:  SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944
TFZ==29.2 PAK=0 LLG=944 TFZ==29.2

   SOLU SPAC P 32 2 1


SG P3212:

 Solution #1 annotation (history):

   SOLU SET  RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5
PAK=0 LLG=350 TFZ==20.5

   SOLU SPAC P 32 1 2


SG P32

SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0
LLG=436 TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0

LLG=1715 TFZ==34.3

   SOLU SPAC P 32


Based on TFZ and LLG, the P32 seems to be best. But I'll also try to
refine and build P32 2 1 latter


On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson 
wrote:

> First - four way twinning is possible but pretty rare for macromolecules
>
> Pointless gives a very useful table of the CC agreement for each possible
> symmetry operator individually.
> In this case with only two molecules in the asymmetric unit you you could
> only have a higher symmetry SG as
> P32 21 P32 12 or P64
>
> These would require as symmetry operators -
> P32 21 - a three fold and a two fold k h -l
> P32 12 - a three fold and a two fold -k -h -l
>
> P64 - a six fold
>
> If the scores for one set are better than the others you probably have
> that SG
>
> However high degrees of twinning can disguise the symmetry scores of
> course..
>
>
>
> On 14 April 2017 at 04:46, Keller, Jacob  wrote:
>
>> 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 
>> 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
>> 

Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services

[Edward Snell]

Dear Elizabeth,


The High-Throughput Crystallization Screening Center and the Hauptman-Woodward 
Medical Research Institute has been operating for over a decade with 
considerable success. There are comprehensive details at http://getacrystal.org 
but basically your samples are screened against a large range of commercially 
available conditions plus some more unique ones. Video microscope images are 
provided over a period of six weeks or longer by request and the imaging also 
includes SONICC (detecting very tiny crystals or crystals in precipitate) and 
UV-TPEF to ensure the sample is protein. There are a large range of analysis 
tools that can be used on the data. Much of the information is provided on the 
link above. Success rates are pretty high and many entries in the PDB have had 
their initial crystal hits there.


I hope this helps, the center does not advertise much but works with a lot of 
laboratories.


Cheers,


Eddie.

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: 
esn...@hwi.buffalo.edu
[https://mail.google.com/mail/u/0/?ui=2=62aec2015e=fimg=15b6914f7fdf6013=0.1=emb=ANGjdJ9g06QLo-D_EH41Fh-YwWWc3MjxJbO2-5rUPyw9xaU5whcTtrcEMgp6IRAWj-flG5nAubbpuMSCQvS1Drhckx7xSgFjo3hKVAHILDzuOysZW5m7BjsDwfq3SJc=w656-h112=1492181842461=15b6914f7fdf6013=1]
Heisenberg was probably here!




From: CCP4 bulletin board  on behalf of Elizabeth Diaz 

Sent: Friday, April 14, 2017 9:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Recommendations for Robotic Crystal Screening Services

All,

I am currently attempting to crystallize a peptide/protein complex and am 
wanting to know about robotic screening services that you would recommend. We 
have done some in house conditions with little luck, and want to broaden our 
search, but that would require going externally for screening. Do you have any 
recommendations for robotic crystal screening services, preferably in the 
United States?

Thank you so much,

Elizabeth Diaz
University of Delaware

Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services

[Parthasarathy Sampathkumar]

Hi Elizabeth,
HWI, University of Buffalo, NY offers high-throughout crystallization
screen. See the link for more information:

http://hwi.buffalo.edu/science/high-throughput-crystallization-center/

Good Luck
Partha
On Fri, Apr 14, 2017 at 9:06 AM Elizabeth Diaz 
wrote:

> All,
>
> I am currently attempting to crystallize a peptide/protein complex and am
> wanting to know about robotic screening services that you would recommend.
> We have done some in house conditions with little luck, and want to broaden
> our search, but that would require going externally for screening. Do you
> have any recommendations for robotic crystal screening services, preferably
> in the United States?
>
> Thank you so much,
>
> Elizabeth Diaz
> University of Delaware
>

Re: [ccp4bb] in crystallo enzymatic activity

[Mark J van Raaij]

I think in his case the WT does not perform the reaction in the crystal, but 
the mutant does.
Haven't heard of examples of this before, but perhaps the WT needs "something" 
to do the reaction, like a conformational change impossible in the crystals, a 
co-factor or something else, perhaps even important for natural regulation.
And the mutant you make somehow lessened this requirement?
Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser .cnb.csic.es/~mjvanraaij 




> On 13 Apr 2017, at 17:35, Bonsor, Daniel  wrote:
> 
> 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] Understanding Biology through Structure, Santa Fe May 13-17

[Terwilliger, Thomas Charles]

Hi Colleagues!


I hope you can come to our symposium on Understanding Biology through Structure 
in Santa Fe this May 13-17!


The Symposium will emphasize interactions between junior and senior 
researchers. We hope that this will present a special opportunity for junior 
researchers to present posters and meet senior scientists, for senior 
researchers to meet the next generation of structural biologists, and for all 
to discuss science in an informal setting.


You can see the full program at 
https://conferences.newmexicoconsortium.org/conferences/ubts_17/program


Understanding Biology through Structure

Santa Fe, NM May 13-17 2017


Speakers


Nucleic acid and protein-nucleic acid complexes

Jamie Cate, University of California, Berkeley
Eva Nogales, University of California, Berkeley
Jody Puglisi, Stanford University
Leemor Joshua-Tor, Cold Spring Harbor Laboratory
Tom Steitz, Yale University
Shigeyuki Yokoyama, RIKEN

Structural Biology of Cell Signaling

Axel Brunger, Stanford University
Sharon Campbell, University of North Carolina
Yvonne Jones, Oxford University
Dorothee Kern, Brandeis University
Cynthia Wolberger, Johns Hopkins University
Hao Wu, Harvard University

Disease and Drug Discovery

Lesa Beamer, University of Missouri
Tom Blundell, University of Cambridge
Elizabeth Goldsmith, UT Southwestern Medical Center
Lynn Howell, University of Toronto
Michael Rossmann, Purdue University
Erica Ollmann Saphire, Scripps Institute
Natalie Strynadka, University of British Columbia
Ian Wilson, Scripps Institute

Crystallographic Methods

Wladek Minor, University of Virginia
Randy Read, University of Cambridge
Jane Richardson, Duke University
Tom Terwilliger, Los Alamos National Laboratory
Isabel Uson, Institute of Molecular Biology, Barcelona
Nadia Zatsepin, University of Arizona

Membrane Protein Structures

Susan Buchanon, National Institutes of Health
Wayne Hendrickson, Columbia University
Robert Stroud, University of California, San Francisco
Nieng Yan, Tsinghua University

Protein folding and Design

David Baker, University of Washington
Ken Dill, Stony Brook University
David Eisenberg, University of California, Los Angeles
Susan Marqusee, University of California, Berkeley
Tobin Sosnick, University of Chicago

Understanding Enzyme Function

Catherine Drennan, Massachusetts Institute of Technology
Andrzej Joachimiak, Argonne National Laboratory
Ci Ji Lim, University of Colorado, Boulder
Ilme Schlichting, Max Planck Institute for Medical Research

All the best,
Tom T
?

?

Re: [ccp4bb] Co-crystal structure of a USP with small molecule inhibitor

[xinhuang...@yahoo.com]

USP not UPS. Sorry about the auto spelling correction feature of my smart (or 
not smart!) phone.
-- Original message--From: 
xinhuang...@yahoo.com<0317accc4341-dmarc-requ...@jiscmail.ac.uk>Date: Fri, 
Apr 14, 2017 7:34 AMTo: CCP4BB@JISCMAIL.AC.UK;Cc: Subject:[ccp4bb] Co-crystal 
structure of a UPS with small molecule inhibitor
Hi, all. 
Could you let me know if there is a co-crystal structure of any UPS in complex 
with a small molecule inhibitor? I cannot seem to find any.
Thanks.

[ccp4bb] Co-crystal structure of a UPS with small molecule inhibitor

[xinhuang...@yahoo.com]

Hi, all. 
Could you let me know if there is a co-crystal structure of any UPS in complex 
with a small molecule inhibitor? I cannot seem to find any.
Thanks.

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

[Eleanor Dodson]

First - four way twinning is possible but pretty rare for macromolecules

Pointless gives a very useful table of the CC agreement for each possible
symmetry operator individually.
In this case with only two molecules in the asymmetric unit you you could
only have a higher symmetry SG as
P32 21 P32 12 or P64

These would require as symmetry operators -
P32 21 - a three fold and a two fold k h -l
P32 12 - a three fold and a two fold -k -h -l

P64 - a six fold

If the scores for one set are better than the others you probably have that
SG

However high degrees of twinning can disguise the symmetry scores of
course..



On 14 April 2017 at 04:46, Keller, Jacob  wrote:

> 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 
> 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
>
>
>
>