[ccp4bb] AW: [ccp4bb] Space group problem
Dear Rain, Maybe a little late, but here are some more comments: 1) What is the deal? For me, one can only know the space group after the structure has been solved. I have seen quite few cases (e.g. twinning, non-crystallographic symmetry etc.) that all programs (XDS, pointless) and statistics would insist on the wrong space group. Since there is a discussion on the space group, the structure has obviously not yet been solved, so why not say something like: most probably hexagonal or maybe trigonal? You could even give statistics for both possibilities. Based on diffraction data alone, it is impossible to determine the space group with certainty (at least for proteins). 2) Higher than expected Rmeas at high resolution also make me suspicious. In such cases I usually compare the statistics of the data processed in P1 vs. the data processed in a higher symmetry space group. If there is a big discrepancy at high resolution, there may be a mundane explanation like radiation damage or anisotropic diffraction, but it might also be that the symmetry which was thought to be crystallographic is in fact non-crystallographic. At low resolution, this symmetry closely matches crystallographic symmetry, but at high resolution, this symmetry deviates more and more resulting in much higher Rfactors. So in fact, you may have a lower symmetry space group with almost crystallographic non-crystallographic symmetry. In your case I would tell the referee that since the structure has not yet been solved it is impossible to determine the exact space group and leave it at that. Best regards, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Rain Field Gesendet: Mittwoch, 7. Mai 2014 21:41 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Space group problem I thought there was a theoretical Rmeas number for I/sigma=2 that is around 50%? In my case, the Rmeas is much higher. Before, we reported P62(4) and got rejected. I'll just change a journal. Hopefully reviewer will not be too picky about that. Thanks to everyone!
Re: [ccp4bb] Space group problem
Hi,
please post the pointless table Analysing rotational symmetry in lattice
group P 6/m m m. This gives a quite clear information about the
presence/absence of rotational symmetry axes.
Look at at the following example
Analysing rotational symmetry in lattice group P 6/m m m
--
!--SUMMARY_BEGIN--
Scores for each symmetry element
Nelmt Lklhd Z-ccCCN RmeasSymmetry operator (in Lattice
Cell)
1 0.936 9.71 0.978676 0.077 identity
2 0.936 9.74 0.97 12213 0.071 *** 2-fold l ( 0 0 1) {-h,-k,l}
3 0.055 1.58 0.16 12097 0.514 2-fold k ( 0 1 0) {-h,h+k,-l}
4 0.056 1.74 0.17 12146 0.495 2-fold h ( 1 0 0) {h+k,-k,-l}
5 0.056 1.73 0.17 12274 0.505 2-fold ( 1-1 0) {-k,-h,-l}
6 0.055 1.61 0.16 12238 0.521 2-fold ( 2-1 0) {h,-h-k,-l}
7 0.056 1.69 0.17 12148 0.495 2-fold (-1 2 0) {-h-k,k,-l}
8 0.056 1.71 0.17 12056 0.511 2-fold ( 1 1 0) {k,h,-l}
9 0.248 6.35 0.64 24331 0.326 3-fold l ( 0 0 1)
{k,-h-k,l}{-h-k,h,l}
10 0.245 6.32 0.63 24629 0.327 6-fold l ( 0 0 1)
{h+k,-h,l}{-k,h+k,l}
Here, the 2-fold symmetry is just as good as the identity, and all the other
symmetries are insignificant, with one exception: there is a pseudo-3fold that
together with the true 2-fold gives a pseudo-6fold. So the true space group is
P2 (or P2_1) but it has 3-fold NCS.
I have seen even more interesting cases, where the CC was 0.8 to 0.9 for some
rotational elements - but even such a seemingly high CC was significantly lower
than the CC for the identity.
So my advice is: look at this table, and compare the CC values with that of the
identity operation.
Two caveats are:
1) a CC may be lower than the identity, due to radiation damage (but that must
then be quite significant)
2) a CC may be high, but still not mean true crystallographic symmetry, if
there is twinning
Finally, a different topic: Rmerge and Rmeas refer to precision of unmerged
data, and their values have no immediate relation to I/sigma, a precision
indicator for the merged data (in this table). So when somehow trying to
compare these values, you are comparing apples to oranges - but this is
meaningless.
HTH,
Kay
On Wed, 7 May 2014 18:26:24 +0100, Rain Field rainfiel...@163.com wrote:
Hi all,
I have a 360 degree data set collect on home beam.
I used XDS to integrate the frames in P1.
I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the
log below.
The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are
much higher than normal at I/sigmaI=2.
I think P1 might be the true space group. But the Rpim reported by aimless
seems high in the high resolution shell. Why is that?
Thanks!
LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM DEGREES)
REINDEXING TRANSFORMATION
CHARACTER LATTICE OF FIT a b c alpha beta gamma
* 31aP 0.0 62.5 82.5 82.6 60.1 89.9 90.0 -1
0 0 0 0 -1 -1 0 0 -1 0 0
* 44aP 0.2 62.5 82.5 82.6 119.9 90.1 90.01
0 0 0 0 1 1 0 0 -1 0 0
* 41mC 0.6 143.1 82.5 62.5 90.0 90.1 90.00
1 -1 0 0 -1 -1 0 -1 0 0 0
* 30mC 0.7 82.5 143.1 62.5 89.9 90.0 90.00
-1 -1 0 0 1 -1 0 1 0 0 0
* 35mP 1.0 82.5 62.5 82.6 90.1 119.9 90.00
-1 -1 0 -1 0 0 0 0 1 0 0
* 40oC 1.2 82.5 143.1 62.5 89.9 90.0 90.00
-1 -1 0 0 -1 1 0 -1 0 0 0
* 20mC 2.6 142.9 82.6 62.5 90.0 90.0 90.10
-2 -1 0 0 0 -1 0 1 0 0 0
* 23oC 3.3 82.6 142.9 62.5 90.0 90.0 89.90
0 1 0 0 -2 -1 0 1 0 0 0
* 25mC 3.3 82.6 142.9 62.5 90.0 90.0 89.90
0 1 0 0 -2 -1 0 1 0 0 0
* 22hP 3.5 82.5 82.6 62.5 90.1 90.0 119.90
1 1 0 0 -1 0 0 1 0 0 0
37mC249.8 176.5 62.5 82.5 90.0 117.8 69.31
-2 0 0 1 0 0 0 0 1 1 0
42oI250.0 62.5 82.5 156.1 90.0 113.6 90.0 -1
0 0 0 0 -1 -1 0 1 -1 1 0
39mC250.6 176.4 62.5 82.6 90.0 117.9 69.21
-2 -2 0 1 0 0 0 0 0 1 0
33mP434.8 62.5 82.5 82.6 119.9 90.1 90.01
0 0 0 0 1 1 0 0 -1 0 0
34mP435.2 62.5 82.6 82.5 119.9 90.0 90.1 -1
0 0 0 0 1 0 0 0 -1 -1 0
32oP435.4 62.5 82.5 82.6 119.9 90.1 90.01
0 0 0 0 1 1 0 0 -1 0 0
21tP437.6 82.5 82.6 62.5 90.1 90.0 119.90
1 1 0 0 -1 0 0 1 0 0 0
P1:
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS
[ccp4bb] AW: [ccp4bb] Space group problem
Dear all, To add another bit to the discussion, I would say that an increase of Rmerge and Rmeas is just expected in such a case, isn't it? According to your tables, in P1, the multiplicity is about 4. In P2, it's about 7. In P3, it's 10. And in P6, it's approaching 20. I would say that this leads us to the now classical problem those statistics have with high multiplicity datasets. Best, Christophe -- Christophe Wirth, PhD Centre for Biological Signalling Studies (bioss) Institute for Biochemistry and Molecular Biology University of Freiburg Stefan-Meier-Str. 17 D-79104 Freiburg, Germany Tel: +49 (0) 761 203 52 77 -- -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Rain Field Gesendet: Mittwoch, 7. Mai 2014 19:26 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Space group problem Hi all, I have a 360 degree data set collect on home beam. I used XDS to integrate the frames in P1. I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the log below. The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are much higher than normal at I/sigmaI=2. I think P1 might be the true space group. But the Rpim reported by aimless seems high in the high resolution shell. Why is that? Thanks! LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM DEGREES) REINDEXING TRANSFORMATION CHARACTER LATTICE OF FIT a b c alpha beta gamma * 31aP 0.0 62.5 82.5 82.6 60.1 89.9 90.0 -1 0 0 0 0 -1 -1 0 0 -1 0 0 * 44aP 0.2 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 * 41mC 0.6 143.1 82.5 62.5 90.0 90.1 90.00 1 -1 0 0 -1 -1 0 -1 0 0 0 * 30mC 0.7 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 1 -1 0 1 0 0 0 * 35mP 1.0 82.5 62.5 82.6 90.1 119.9 90.00 -1 -1 0 -1 0 0 0 0 1 0 0 * 40oC 1.2 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 -1 1 0 -1 0 0 0 * 20mC 2.6 142.9 82.6 62.5 90.0 90.0 90.10 -2 -1 0 0 0 -1 0 1 0 0 0 * 23oC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 25mC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 22hP 3.5 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 37mC249.8 176.5 62.5 82.5 90.0 117.8 69.31 -2 0 0 1 0 0 0 0 1 1 0 42oI250.0 62.5 82.5 156.1 90.0 113.6 90.0 -1 0 0 0 0 -1 -1 0 1 -1 1 0 39mC250.6 176.4 62.5 82.6 90.0 117.9 69.21 -2 -2 0 1 0 0 0 0 0 1 0 33mP434.8 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 34mP435.2 62.5 82.6 82.5 119.9 90.0 90.1 -1 0 0 0 0 1 0 0 0 -1 -1 0 32oP435.4 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 21tP437.6 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 P1: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.4545001165 1288 90.5% 2.2% 2.4% 4498 51.19 2.6%99.9*100.8381030 6.2771741844 1872 98.5% 3.1% 3.0% 7173 35.92 3.6%99.9*11* 0.9061671 5.2090092288 2324 98.5% 4.5% 4.3% 9008 26.88 5.2%99.8* 40.8352168 4.55 105972686 2744 97.9% 5.2% 5.2% 10595 22.65 6.0%99.7*-20.7762557 4.09 120513051 3147 96.9% 7.5% 7.5% 12048 16.93 8.7%99.4* 20.7792915 3.75 127403226 3342 96.5% 14.8% 14.5% 127389.37 17.1%98.2* 10.7603078 3.48 143443631 3738 97.1% 22.2% 22.2% 143426.36 25.7%95.8* 00.7853471 3.26 150793813 3948 96.6% 47.3% 48.5% 150762.99 54.8%83.7*-10.7143655 3.08 140883797 4242 89.5% 99.7%105.6% 139451.30116.0
Re: [ccp4bb] AW: [ccp4bb] Space group problem
On Thu, 8 May 2014 16:56:01 +0200, Christophe Wirth christophe.wi...@biochemie.uni-freiburg.de wrote: Dear all, To add another bit to the discussion, I would say that an increase of Rmerge and Rmeas is just expected in such a case, isn't it? According to your tables, in P1, the multiplicity is about 4. In P2, it's about 7. In P3, it's 10. And in P6, it's approaching 20. I would say that this leads us to the now classical problem those statistics have with high multiplicity datasets. Rmeas does not have this problem, only Rmerge has it! But there are other factors which produce higher (and more realistic!) Rmeas in the higher-symmetry spacegroup, like radiation damage and absorption or other systematic differences that can not be removed by scaling. In other words: in an ideal experiment Rmeas should be the same for the correct space group and its sub-groups. In a real experiment, however, Rmeas in a high-symmetry space group sees the differences resulting from systematic errors, whereas Rmeas in a low-symmetry space group often does not see it - simply because it does not compare those reflections which suffer from the error. best, Kay Best, Christophe -- Christophe Wirth, PhD Centre for Biological Signalling Studies (bioss) Institute for Biochemistry and Molecular Biology University of Freiburg Stefan-Meier-Str. 17 D-79104 Freiburg, Germany Tel: +49 (0) 761 203 52 77 -- -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Rain Field Gesendet: Mittwoch, 7. Mai 2014 19:26 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Space group problem Hi all, I have a 360 degree data set collect on home beam. I used XDS to integrate the frames in P1. I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the log below. The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are much higher than normal at I/sigmaI=2. I think P1 might be the true space group. But the Rpim reported by aimless seems high in the high resolution shell. Why is that? Thanks! LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM DEGREES) REINDEXING TRANSFORMATION CHARACTER LATTICE OF FIT a b c alpha beta gamma * 31aP 0.0 62.5 82.5 82.6 60.1 89.9 90.0 -1 0 0 0 0 -1 -1 0 0 -1 0 0 * 44aP 0.2 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 * 41mC 0.6 143.1 82.5 62.5 90.0 90.1 90.00 1 -1 0 0 -1 -1 0 -1 0 0 0 * 30mC 0.7 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 1 -1 0 1 0 0 0 * 35mP 1.0 82.5 62.5 82.6 90.1 119.9 90.00 -1 -1 0 -1 0 0 0 0 1 0 0 * 40oC 1.2 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 -1 1 0 -1 0 0 0 * 20mC 2.6 142.9 82.6 62.5 90.0 90.0 90.10 -2 -1 0 0 0 -1 0 1 0 0 0 * 23oC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 25mC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 22hP 3.5 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 37mC249.8 176.5 62.5 82.5 90.0 117.8 69.31 -2 0 0 1 0 0 0 0 1 1 0 42oI250.0 62.5 82.5 156.1 90.0 113.6 90.0 -1 0 0 0 0 -1 -1 0 1 -1 1 0 39mC250.6 176.4 62.5 82.6 90.0 117.9 69.21 -2 -2 0 1 0 0 0 0 0 1 0 33mP434.8 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 34mP435.2 62.5 82.6 82.5 119.9 90.0 90.1 -1 0 0 0 0 1 0 0 0 -1 -1 0 32oP435.4 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 21tP437.6 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 P1: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.4545001165 1288 90.5% 2.2% 2.4% 4498 51.19 2.6%99.9*100.8381030 6.2771741844 1872 98.5% 3.1% 3.0% 7173 35.92 3.6%99.9*11* 0.9061671 5.2090092288 2324 98.5% 4.5% 4.3% 9008 26.88
[ccp4bb] Space group problem
Hi all, I have a 360 degree data set collect on home beam. I used XDS to integrate the frames in P1. I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the log below. The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are much higher than normal at I/sigmaI=2. I think P1 might be the true space group. But the Rpim reported by aimless seems high in the high resolution shell. Why is that? Thanks! LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM DEGREES) REINDEXING TRANSFORMATION CHARACTER LATTICE OF FIT a b c alpha beta gamma * 31aP 0.0 62.5 82.5 82.6 60.1 89.9 90.0 -1 0 0 0 0 -1 -1 0 0 -1 0 0 * 44aP 0.2 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 * 41mC 0.6 143.1 82.5 62.5 90.0 90.1 90.00 1 -1 0 0 -1 -1 0 -1 0 0 0 * 30mC 0.7 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 1 -1 0 1 0 0 0 * 35mP 1.0 82.5 62.5 82.6 90.1 119.9 90.00 -1 -1 0 -1 0 0 0 0 1 0 0 * 40oC 1.2 82.5 143.1 62.5 89.9 90.0 90.00 -1 -1 0 0 -1 1 0 -1 0 0 0 * 20mC 2.6 142.9 82.6 62.5 90.0 90.0 90.10 -2 -1 0 0 0 -1 0 1 0 0 0 * 23oC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 25mC 3.3 82.6 142.9 62.5 90.0 90.0 89.90 0 1 0 0 -2 -1 0 1 0 0 0 * 22hP 3.5 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 37mC249.8 176.5 62.5 82.5 90.0 117.8 69.31 -2 0 0 1 0 0 0 0 1 1 0 42oI250.0 62.5 82.5 156.1 90.0 113.6 90.0 -1 0 0 0 0 -1 -1 0 1 -1 1 0 39mC250.6 176.4 62.5 82.6 90.0 117.9 69.21 -2 -2 0 1 0 0 0 0 0 1 0 33mP434.8 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 34mP435.2 62.5 82.6 82.5 119.9 90.0 90.1 -1 0 0 0 0 1 0 0 0 -1 -1 0 32oP435.4 62.5 82.5 82.6 119.9 90.1 90.01 0 0 0 0 1 1 0 0 -1 0 0 21tP437.6 82.5 82.6 62.5 90.1 90.0 119.90 1 1 0 0 -1 0 0 1 0 0 0 P1: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.4545001165 1288 90.5% 2.2% 2.4% 4498 51.19 2.6%99.9*100.8381030 6.2771741844 1872 98.5% 3.1% 3.0% 7173 35.92 3.6%99.9*11* 0.9061671 5.2090092288 2324 98.5% 4.5% 4.3% 9008 26.88 5.2%99.8* 40.8352168 4.55 105972686 2744 97.9% 5.2% 5.2% 10595 22.65 6.0%99.7*-20.7762557 4.09 120513051 3147 96.9% 7.5% 7.5% 12048 16.93 8.7%99.4* 20.7792915 3.75 127403226 3342 96.5% 14.8% 14.5% 127389.37 17.1%98.2* 10.7603078 3.48 143443631 3738 97.1% 22.2% 22.2% 143426.36 25.7%95.8* 00.7853471 3.26 150793813 3948 96.6% 47.3% 48.5% 150762.99 54.8%83.7*-10.7143655 3.08 140883797 4242 89.5% 99.7%105.6% 139451.30116.0%58.6*-20.6363133 total 99582 25501 26645 95.7% 7.7% 7.9% 99423 14.49 9.0%99.9* 10.765 23678 P2: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.454499 638 711 89.7% 2.5% 2.7% 4497 63.58 2.8% 100.0*16* 0.892 534 6.277172 986 993 99.3% 3.5% 3.5% 7172 46.06 3.7%99.9*100.948 872 5.2089631202 1213 99.1% 4.9% 4.9% 8963 35.19 5.3%99.9* 20.8491101 4.55 105981413 1427 99.0% 5.8% 5.9% 10598 29.79
Re: [ccp4bb] Space group problem
additional info: If I let xds go through, it will choose P6. actually pointless suggest P62/P64. The thing is the Rmeas and Rmerge are significantly higher for P2/P3/P6 than P1, especially the highest shell. That indicates those higher symmetry ones are not the choice, it that right? (Actually, this is also the reviewer's question) P6 log from xds: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.454512 216 239 90.4% 3.1% 3.3% 4511 95.55 3.2% 100.0*41* 1.124 182 6.277208 330 330 100.0% 4.0% 4.0% 7208 72.47 4.1% 100.0*22* 1.211 293 5.208925 401 405 99.0% 5.4% 5.4% 8925 56.60 5.5%99.9*120.965 368 4.55 10585 470 474 99.2% 6.3% 6.5% 10585 48.76 6.5% 100.0* 30.841 436 4.09 11898 529 542 97.6% 9.1% 9.0% 11898 37.80 9.3%99.9*-60.809 495 3.75 12425 550 573 96.0% 16.9% 16.7% 12425 22.09 17.3%99.7* 10.855 519 3.48 14344 638 639 99.8% 26.3% 26.1% 14344 14.57 26.9%99.3*-30.783 601 3.26 15001 668 671 99.6% 53.6% 56.1% 150017.15 54.8%96.9*-20.745 633 3.08 14177 704 725 97.1% 114.8%124.6% 141652.95117.7%87.5*-30.632 637 total 990754506 4598 98.0% 9.3% 9.5% 99062 30.73 9.5% 100.0* 10.8334164
Re: [ccp4bb] Space group problem
I thought there was a theoretical Rmeas number for I/sigma=2 that is around 50%? In my case, the Rmeas is much higher. Before, we reported P62(4) and got rejected. I'll just change a journal. Hopefully reviewer will not be too picky about that. Thanks to everyone!
Re: [ccp4bb] Space group problem
It is definitely P6something or P3something with a twinning about the unique axis. The differences in merging statistics between P2 P3 and P6 are not very significant in my opinion. On May 7, 2014, at 1:16 PM, Rain Field rainfiel...@163.com wrote: additional info: If I let xds go through, it will choose P6. actually pointless suggest P62/P64. The thing is the Rmeas and Rmerge are significantly higher for P2/P3/P6 than P1, especially the highest shell. That indicates those higher symmetry ones are not the choice, it that right? (Actually, this is also the reviewer's question) P6 log from xds: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.454512 216 239 90.4% 3.1% 3.3% 4511 95.55 3.2% 100.0*41* 1.124 182 6.277208 330 330 100.0% 4.0% 4.0% 7208 72.47 4.1% 100.0*22* 1.211 293 5.208925 401 405 99.0% 5.4% 5.4% 8925 56.60 5.5%99.9*120.965 368 4.55 10585 470 474 99.2% 6.3% 6.5% 10585 48.76 6.5% 100.0* 30.841 436 4.09 11898 529 542 97.6% 9.1% 9.0% 11898 37.80 9.3%99.9*-60.809 495 3.75 12425 550 573 96.0% 16.9% 16.7% 12425 22.09 17.3%99.7* 10.855 519 3.48 14344 638 639 99.8% 26.3% 26.1% 14344 14.57 26.9%99.3*-30.783 601 3.26 15001 668 671 99.6% 53.6% 56.1% 150017.15 54.8%96.9*-20.745 633 3.08 14177 704 725 97.1% 114.8%124.6% 141652.95117.7%87.5*-30.632 637 total 990754506 4598 98.0% 9.3% 9.5% 99062 30.73 9.5% 100.0* 10.8334164
Re: [ccp4bb] Space group problem
Hello Rain Field, I third Felix and Craig, there is nothing unusual about the statistics table you present. The data look pretty good and I would assume P6_something and integrate further than 3.1A. Regards, Tim On 05/07/2014 08:16 PM, Rain Field wrote: additional info: If I let xds go through, it will choose P6. actually pointless suggest P62/P64. The thing is the Rmeas and Rmerge are significantly higher for P2/P3/P6 than P1, especially the highest shell. That indicates those higher symmetry ones are not the choice, it that right? (Actually, this is also the reviewer's question) P6 log from xds: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 8.454512 216 239 90.4% 3.1% 3.3% 4511 95.55 3.2% 100.0*41* 1.124 182 6.277208 330 330 100.0% 4.0% 4.0% 7208 72.47 4.1% 100.0*22* 1.211 293 5.208925 401 405 99.0% 5.4% 5.4% 8925 56.60 5.5%99.9*120.965 368 4.55 10585 470 474 99.2% 6.3% 6.5% 10585 48.76 6.5% 100.0* 30.841 436 4.09 11898 529 542 97.6% 9.1% 9.0% 11898 37.80 9.3%99.9*-60.809 495 3.75 12425 550 573 96.0% 16.9% 16.7% 12425 22.09 17.3%99.7* 10.855 519 3.48 14344 638 639 99.8% 26.3% 26.1% 14344 14.57 26.9%99.3*-30.783 601 3.26 15001 668 671 99.6% 53.6% 56.1% 150017.15 54.8%96.9*-20.745 633 3.08 14177 704 725 97.1% 114.8%124.6% 141652.95117.7%87.5*-30.632 637 total 990754506 4598 98.0% 9.3% 9.5% 99062 30.73 9.5% 100.0* 10.8334164 -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Space group problem?
Dear Nazia, My first suggestion would be to take your crystals to a synchrotron. With the same crystals, you may get a precious 0.5 Å resolution more. Concerning the processing, I agree with Jürgen that the beam center is the most frequent culprit. The second thing to do is to make sure that you do not impose any space group and let your data processing program determine the space group, which may be very different from what you expect. Next you should inspect your images for potential problems: ice/salt rings, very high mosaicity, smeared or blurred spots, bad zones etc. With poorly diffracting crystals, processing using default values may not work and you may have to fine-tune your processing parameters. Best would be to find a local expert, otherwise you should provide the bulletin board with more details like processing programs tried, expected cell parameters, assessment of the diffraction pattern etc. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nazia Nasir Phd2009,ProteinCrystall.Lab Gesendet: Dienstag, 1. April 2014 20:00 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] Space group problem? Dear Jurgen, The beam position is fine. we have collected many data sets before and after this data. Moreover, we the Technical scientist always checks the beam position before we mount the crystals. Thanks On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch jbos...@jhu.edumailto:jbos...@jhu.edu wrote: check your beam position .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.inmailto:nazia.nasi...@nii.ac.in wrote: Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edumailto:c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Chen Zhao Gesendet: Montag, 31. März 2014 23:46 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Space group
Re: [ccp4bb] Space group problem?
Dear Chen, how sure are you that your crystals contain the protein of interest? Repeatedly washing harversted crystals in crystal stabilization solution followed by SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver staining) or western blotting can give a pretty conclusive answer. In our group, SDS-PAGE followed by Silver-staining is done routinely and in most cases leads to conclusive results. Here is a summary of the protocol: - select a drop containing substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of motherliquor containing a 10% higher concentration of precipitant). - transfer all the crystalline material from the drop into the PCR-tube using a pipet (You can use stabilization buffer from the PCR tube to collect all crystals). One can also use a cryo-loop to harvest the crystals if they are large enough to allow efficient harvesting. - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope to make sure they are at the bottom of the PCR-tube. - remove as much of the supernatant as you can using a pipet making sure not to remove the crystals. Then add crystal stabilization buffer to wash the crystals, and centrifuge again. - repeat this washing procedure a few times (typicaly 3-4 times). - after the final washing step, centrifugation and removal of the supernatant, add Laemmli-buffer to the crystals and use this sample for loading onto the SDS-PAGE gel. - include a positive control (e.g. solubilize another drop directly in Laemmli-buffer) and a negative control (final washing buffer). Including a pre-crystallization sample of your protein as a control is also recommended, to control for the integrity of the protein under crystallization conditions. - use silver staining to visualize the protein. --- best regards Savvas On 31 Mar 2014, at 23:48, Chen Zhao c.z...@yale.edu wrote: BTW, I forgot to mention that phenix.autosol also gave similar result. On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
Re: [ccp4bb] Space group problem?
Dear Savvas, Thank you for your reply and your nice protocol. I am also worried of this problem so I have already run my crystals on the gel for several times. The result is so clean that you can hardly draw any other conclusions except that the crystals are made up of the full-length molecule. But possibility remains regarding this concern if my molecule of interest is not in crystalline state but other molecules that cannot be stained are. Best, Chen On Tue, Apr 1, 2014 at 3:30 AM, Savvas Savvides savvas.savvi...@ugent.bewrote: Dear Chen, how sure are you that your crystals contain the protein of interest? Repeatedly washing harversted crystals in crystal stabilization solution followed by SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver staining) or western blotting can give a pretty conclusive answer. In our group, SDS-PAGE followed by Silver-staining is done routinely and in most cases leads to conclusive results. Here is a summary of the protocol: - select a drop containing substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of motherliquor containing a 10% higher concentration of precipitant). - transfer all the crystalline material from the drop into the PCR-tube using a pipet (You can use stabilization buffer from the PCR tube to collect all crystals). One can also use a cryo-loop to harvest the crystals if they are large enough to allow efficient harvesting. - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope to make sure they are at the bottom of the PCR-tube. - remove as much of the supernatant as you can using a pipet making sure not to remove the crystals. Then add crystal stabilization buffer to wash the crystals, and centrifuge again. - repeat this washing procedure a few times (typicaly 3-4 times). - after the final washing step, centrifugation and removal of the supernatant, add Laemmli-buffer to the crystals and use this sample for loading onto the SDS-PAGE gel. - include a positive control (e.g. solubilize another drop directly in Laemmli-buffer) and a negative control (final washing buffer). Including a pre-crystallization sample of your protein as a control is also recommended, to control for the integrity of the protein under crystallization conditions. - use silver staining to visualize the protein. --- best regards Savvas On 31 Mar 2014, at 23:48, Chen Zhao c.z...@yale.edu wrote: BTW, I forgot to mention that phenix.autosol also gave similar result. On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
[ccp4bb] AW: [ccp4bb] Space group problem?
Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Chen Zhao Gesendet: Montag, 31. März 2014 23:46 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Chen Zhao *Gesendet:* Montag, 31. März 2014 23:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
Dear Jurgen, The beam position is fine. we have collected many data sets before and after this data. Moreover, we the Technical scientist always checks the beam position before we mount the crystals. Thanks On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch jbos...@jhu.edu wrote: check your beam position .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.in wrote: Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Chen Zhao *Gesendet:* Montag, 31. März 2014 23:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Chen Zhao *Gesendet:* Montag, 31. März 2014 23:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
[ccp4bb] Space group problem?
Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
Re: [ccp4bb] Space group problem?
BTW, I forgot to mention that phenix.autosol also gave similar result. On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
[ccp4bb] space group problem
Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 ==
Re: [ccp4bb] space group problem
Dear Junyu, it looks to me like you encounter a classical monoclinic feature: one can index monoclinic always in two ways origin | V A' -- A \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / // C C' One cell (A,B,C) has B coming towards you and the other (A',B',C') has B' pointing away from you. The two axes A and A' have identical length as have B and B'. But C' is the diagonal in the AC-plane. In your case you can just swap the A and C axis of the C2 (to follow the above picture) and then calculate the C' (diagonal) to 136.8. So to summarize: these are identical cells - just different choice of axes (and nothing to do with the C2 versus P2 choice ... I think). Cheers Clemens On Thu, May 01, 2008 at 12:03:16PM -0400, Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] space group problem
What is the Rmerge in each case? -James Holton MAD Scientist Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 ==
Re: [ccp4bb] space group problem
Clemens is right of course. If you ignore the lattice centring in C2, the cells are the same. I was however under the impression that auto-indexing goes via the primitive cell. Which makes the two solutions unique. Ignoring the possible lattice translation in P2 will show up in a Patterson function (at 1/2,1/2,0 i think) . The lattice translation might of course be a pseudo translation. In the C2 case, you would miss weak reflections if P2 would be the right answer. P 2008/5/1 Clemens Vonrhein [EMAIL PROTECTED]: Dear Junyu, it looks to me like you encounter a classical monoclinic feature: one can index monoclinic always in two ways origin | V A' -- A \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / // C C' One cell (A,B,C) has B coming towards you and the other (A',B',C') has B' pointing away from you. The two axes A and A' have identical length as have B and B'. But C' is the diagonal in the AC-plane. In your case you can just swap the A and C axis of the C2 (to follow the above picture) and then calculate the C' (diagonal) to 136.8. So to summarize: these are identical cells - just different choice of axes (and nothing to do with the C2 versus P2 choice ... I think). Cheers Clemens On Thu, May 01, 2008 at 12:03:16PM -0400, Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] space group problem
Dear all, Thanks for all the advices. I am especially grateful to Dr. Clemens Vonrhein, now I am clear about the relationship between this two choices. Dr. Zwart raises another interesting point, which of course is my major concern. C2 will have the advantage for phasing, since it has a smaller ASU; but incomplete data won't help. Can I get more education on this? Thanks a lot, Junyu On May 1, 2008, at 1:07 PM, Peter Zwart wrote: Clemens is right of course. If you ignore the lattice centring in C2, the cells are the same. I was however under the impression that auto-indexing goes via the primitive cell. Which makes the two solutions unique. Ignoring the possible lattice translation in P2 will show up in a Patterson function (at 1/2,1/2,0 i think) . The lattice translation might of course be a pseudo translation. In the C2 case, you would miss weak reflections if P2 would be the right answer. P 2008/5/1 Clemens Vonrhein [EMAIL PROTECTED]: Dear Junyu, it looks to me like you encounter a classical monoclinic feature: one can index monoclinic always in two ways origin | V A' -- A \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / // C C' One cell (A,B,C) has B coming towards you and the other (A',B',C') has B' pointing away from you. The two axes A and A' have identical length as have B and B'. But C' is the diagonal in the AC-plane. In your case you can just swap the A and C axis of the C2 (to follow the above picture) and then calculate the C' (diagonal) to 136.8. So to summarize: these are identical cells - just different choice of axes (and nothing to do with the C2 versus P2 choice ... I think). Cheers Clemens On Thu, May 01, 2008 at 12:03:16PM -0400, Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net - = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 ==
Re: [ccp4bb] space group problem
pseudo C2 centering in P21 will result in C2 systematic absenses being weak. you can check this with e.g. dataman (parity check of even and odd reflections of that type). and you will have that native patterson peak as mentioned. this will make life bit more complicated of course since large portion of the data maybe quite weak. also if its really P21 but because of some reason you dont count the weaker reflections in indexing that will lead you to _misindex_ the data in C2 and you are missing the weak data that would tell you some details about it in realite _differs_ from C2. (it is not a matter of incompleteness in either space group/lattice per se.) -i believe this was the point..? hth, -tommi Quoting Junyu Xiao [EMAIL PROTECTED]: Dear all, Thanks for all the advices. I am especially grateful to Dr. Clemens Vonrhein, now I am clear about the relationship between this two choices. Dr. Zwart raises another interesting point, which of course is my major concern. C2 will have the advantage for phasing, since it has a smaller ASU; but incomplete data won't help. Can I get more education on this? Thanks a lot, Junyu On May 1, 2008, at 1:07 PM, Peter Zwart wrote: Clemens is right of course. If you ignore the lattice centring in C2, the cells are the same. I was however under the impression that auto-indexing goes via the primitive cell. Which makes the two solutions unique. Ignoring the possible lattice translation in P2 will show up in a Patterson function (at 1/2,1/2,0 i think) . The lattice translation might of course be a pseudo translation. In the C2 case, you would miss weak reflections if P2 would be the right answer. P 2008/5/1 Clemens Vonrhein [EMAIL PROTECTED]: Dear Junyu, it looks to me like you encounter a classical monoclinic feature: one can index monoclinic always in two ways origin | V A' -- A \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / \ /\ / \ / \ / // C C' One cell (A,B,C) has B coming towards you and the other (A',B',C') has B' pointing away from you. The two axes A and A' have identical length as have B and B'. But C' is the diagonal in the AC-plane. In your case you can just swap the A and C axis of the C2 (to follow the above picture) and then calculate the C' (diagonal) to 136.8. So to summarize: these are identical cells - just different choice of axes (and nothing to do with the C2 versus P2 choice ... I think). Cheers Clemens On Thu, May 01, 2008 at 12:03:16PM -0400, Junyu Xiao wrote: Hi all, We run into an interesting space group problem. The same diffraction image can be either indexed into space group C2, with a=145, b=44, c=67, and beta=110.5; or space group P2 (should be P21 after scaling), with a=67, b=44, c=136, and beta=96.8. Both are refined ok during index. These two must somehow be related. Can anyone give some comments on that? Thanks a lot, Junyu = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net - = Junyu Xiao Department of Biological Chemistry, University of Michigan Lab address: 3163 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue Ann Arbor, MI, 48109-2216 Phone: 734-615-2078 == -- Tommi Kajander, Ph.D.
