### Re: [ccp4bb] low-resolution data and SG

Dear SDY, It is impossible to deduct from data statistics alone the difference between e.g. P43 21 2 and P41 21 2. Also with weak data like you have, a lot of artifacts may arise due to (weak) ice rings, intensity from neighboring strong reflections getting into the integration boxes of weak reflections, spurious reflections due to contaminating salt microcrystals etc.etc.etc. What you need to do is to integrate your data in the basic point group: P4 or P422, depending how sure you are about the additional twofold, and then run Phaser with the SGALTERNATIVE ALL option, so it will check all possible space groups (P41 2 2, P41 21 2, P42 2 2, P42 21 2 etc.). I am pretty sure you will find that the space group which will come out then will not be the P43 21 2 you have assumed right now and that refinement in this space group will solve your problem. Since with your current solution, you will have most of your symmetries correct, you still can get Rfactors in the 30-40% range like you observe. If you wish, after you found the correct space group with Phaser, you could reprocess your data using this correct space group. Best regards, Herman Schreuder From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of SD Y Sent: Sunday, November 04, 2012 4:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] low-resolution data and SG Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. !--[if !supportLists]--1. !--[endif]--I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 !--[if !supportLists]--2. !--[endif]--When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 !--[if !supportLists]--3. !--[endif]--I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it's right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use

### Re: [ccp4bb] low-resolution data and SG

There are several questions here. 1) What is the point Group - P4 or P422. I find the pointless statistics which check all symmetry operators singly useful. If the 2 fold kh-l gives about the same CC as the 4 fold operators k-hl -h -k l -k h l then you probably do have point group P422. ( But check the twinning graph to see if twinning is likely - twinning in this space group can mimic the 2 fold k,h,-l ) and if it is present you should work in point group P4. 2) What is the space group? The absences make it look like P42 21 2 or P 43 21 2 except for the rogues 00 19 and 00 20 which maybe are just that - rogues - see Jims suggestions..) But do you have a non-crystallographic translation? this can be found by a native patterson peak search, and is part of the truncate output. If there IS a peak at x,y,z=1/4 say then that might mislead you be producing weak reflections along 0 0 l axis. 3) Find the MR solution - and that is easiest done using the Phaser command test alterspacegroups.. Eleanor On 4 Nov 2012, at 15:03, SD Y wrote: Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. 1. I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 2. When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 3. I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it’s right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this opportunity to learn refmac well. So could you please let me know if my doubt is right regarding SG and how do I troubleshoot. Thanks SDY

### Re: [ccp4bb] low-resolution data and SG

-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear SDY, if you can see extra density after MR into which you can even build or correct the model it is a good sign your chose the correct space group. Check the geometry of your model. I suppose it is very distorted - a matrix weight of 0.03 sounds high for 3.4A data - You can go down by a factor of 10 at least. You may need to run refmac for many cycles - I have used 200-300 cycles with weight matrix 0.001 and the LL would still not converge. Regards, Tim On 11/04/2012 04:03 PM, SD Y wrote: Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. 1. I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 2. When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 3. I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it’s right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this opportunity to learn refmac well. So could you please let me know if my doubt is right regarding SG and how do I troubleshoot. Thanks SDY - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQlpsuUxlJ7aRr7hoRAgUMAKCJNhlDW4q2Lgmer4lZJoi+GpxDmACg9sRW a5HeDN5HHK/Wdy1sEY+9vbE= =aefJ -END PGP SIGNATURE-

### Re: [ccp4bb] low-resolution data and SG

This looks like an output from SCALEPACK. Unfortunately, one has no way to know from the output if 21 and 90 are strong intensities or not. One cannot go by the I/sigmaI alone. For example, suppose there is thermal diffuse scatter at these positions or perhaps there is a cosmic ray or radioactive decay (zinger) or a spot from a split crystal or the tail of a nearby streaky spot or other error during integrating of these Bragg reflections. I recommend you look at (a) neighboring reflections to get a sense of what a strong reflection value is. Maybe it is 20,000 or more so that 90 would be a weak reflection, and (b) the raw image itself at these reflection positions to see what the actual appearance of the pixel values in these positions are. Jim ... 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 ...