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 <kell...@janelia.hhmi.org>
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 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> 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 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Alex 
Lee
Sent: Friday, April 14, 2017 11:50 AM

To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto: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 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 11:32 PM

To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto: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 
<kell...@janelia.hhmi.org<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<mailto:alexlee198...@gmail.com>]
Sent: Thursday, April 13, 2017 9:08 PM
To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto: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 
<kell...@janelia.hhmi.org<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.<http://journals.iucr.org/d> (2017). 
D73<http://journals.iucr.org/d/contents/backissues.html>, 22-31
https://doi.org/10.1107/S2059798316019318

Jacob

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of 
Eleanor Dodson
Sent: Thursday, April 13, 2017 3:11 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto: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 
<r.joos...@nki.nl<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<mailto:alexlee198...@gmail.com>
Verzonden: donderdag 13 april 2017 19:19
Aan: CCP4BB@JISCMAIL.AC.UK<mailto: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.










Reply via email to