Dear Napo,

For me this looks like a correct treatment of a twinned crystal. What more do 
you want/expect?


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Napoleao 
Fonseca Valadares
Gesendet: Mittwoch, 30. November 2016 08:01
Betreff: [ccp4bb] To win or not twinning?

Dear CCP4ers,

I'd like to kindly ask your advice. Sorry for the long e-mail.

I have crystals of a 12.3 KDa protein that grow in hexagon-like patterns, link 
for the crystal image:

XDS, Phenix and Pointless always suggest that the data sets for these crystals 
belong to the space group P622. However, Phenix, Phaser and Pointless indicate 
that twinning is present.

"Bad looking" diffraction images, diffracted to 1.6 A (collected 6 months ago):

Best data set, diffracted to 2.01 A (collected a month ago):

The second data set present better looking images, a better XDS ISa value 
(around 24) and diffracted to 2.2 A. The "bad looking" data set diffracted to 
1.6 A, but I decided to stop working with it (XDS ISa around 10).

There is a template with 60% identity, I used XDS to try to process the data in 
all trigonal/hexagonal space groups from P3 to P6(3)22, and spend a lot of time 
trying molecular replacement procedures in Phaser and Morda, and refining the 
candidate solutions. Used Zanuda too, trying to figure out the space group (and 
read as much as possible in this CCP4 list looking for similar cases).

Unit cells and typical MR results:

P1: 56.430   77.718   77.673 119.99  89.99  89.97
    SOLU SET  RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311 TFZ==18.4 
    TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751 TFZ==64.6

P3: 77.675   77.675   56.409  90.00  90.00 120.00
    RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5

P6: 77.534   77.534   92.986  90.00  90.00 120.00
    RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7

P6(3): 77.675   77.675   56.409  90.00  90.00 120.00
       RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6

P622: 77.660   77.660   56.400  90.00  90.00 120.00
      RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8

In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric unit, and 
in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, in P1 the ASU looks 
like two superposed hexagonal donuts formed by 6 molecules each.
Refining in P1, without adding waters or TLS, yield r_work = 0.2964 and r_free 
= 0.3428, and it is hard to decrease these values.
Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, but looks 
like it's not getting any better than this.
Refining in P6 yields horrible r_free values (>0.50).

Trying to refine MR solutions in any other other space groups yield Rfree 0.41 
or more.

If in the P3 space group I use the twin operator -k,-h,-l (estimated twin 
fraction of 0.490) suggested by Phenix Xtriage, the values miraculously go down 
to r_work = 0.1923 r_free = 0.2202, without waters (r_free without using a twin 
law = 0.3399). Adding 120 water molecules and doing some refining yields 0.1711 
r_free = 0.1935 (the asymmetric unit contains 420 residues and the resolution 
is 2.01 A).
I've been reading about twinning refinement and how it can drop the Rvalues, 
but from what I understood if I use it improperly I may compromise the 
refinement quality.

I would like advice on:
1 - References that can teach how to look at protein diffraction images and 
understand what I am seeing. The basics like recognizing bad data and what 
usually leads to deformity in the spots (for example, elliptical or duplicated) 
would be of great help.

2 - Should I look for other space groups? What else could be tried? Is this a 
case where a twin law should be used in the refinement? If yes, what can I do 
to confirm the need for a twin law in the refinement?

Thank you all in advance.

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