Dear Napo, I imagine this article, although rather wide ranging in its content, will be of assistance:- http://journals.iucr.org/d/issues/2005/06/00/ic5050/ More generally you might consult Chayen, Helliwell and Snell "Macromolecular Crystallisation and Crystal Perfection" Published by OUP and IUCr. [Apologies to CCP4bb colleagues as this involves a commercial product ie my own book.] All best wishes, John
On Wed, Nov 30, 2016 at 7:01 AM, Napoleao Fonseca Valadares < n...@ifsc.usp.br> wrote: > 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: > http://fullonline.org/science/cryst01.jpg > > 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): > http://fullonline.org/science/dataset1_image37.png > http://fullonline.org/science/dataset1_image7.png > > Best data set, diffracted to 2.01 A (collected a month ago): > http://fullonline.org/science/dataset2_image51.png > > 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. > Regards, > Napo > -- Professor John R Helliwell DSc
