Print

Print
I second Jürgen's suggestion of fixing the distance -- this is often quite helpful when dealing with difficult datasets, at least in my experience 

And this goes without saying, but also double check your beamcenter and try masking the beamstop (using UNTRUSTED_ELLIPSE) if you haven't done so already



On Jul 17, 2012, at 12:17 AM, "Bosch, Juergen" <[log in to unmask]> wrote:

I'd run 
INTEGRATE(REFINE)=CELL
CORRECT(REFINE)=CELL

and fixing your distance first. Then once XDS is done copy GXPARM.XDS to XPARM.XDS and enter those refined values into your XDS.INP script. Once you have a stable cell you can refine the distance and later fix that one. Moving distance is a bit worrying unless you experience an earthquake while collecting. It's more an indication that your data is not good enough to provide a stable refinement of all parameters at once. @Kai feel free to correct me here.

For next time you could ask the following questions:
a) am I using the whole detector area ?
b) do I really need such high resolution for what I want ?
c) collecting oscillations less then 1/3 of your mosaicity does not gain you much in terms of signal/noise you just spent more time collecting and perhaps frying your crystal before obtaining a complete dataset
d) for HA less is more collect lower res 4Å for example but quick and reliable. Since you can pull the detector further back you won't run into overlap issues (but check it out with testgen in Mosflm or iMosflm via GUI)

I think you are doing very well with what you have in terms of data.

Jürgen

On Jul 17, 2012, at 12:04 AM, Jason Busby wrote:

Hi,

Ok, IDXREF.LP shows that it was only using 1-262.  I tried running COLSPOT and IDXREF again, and it picks the same unit cell.

Pointless picks Pmmm and picks 2 definite screw axes, and one possible (p ~0.5), so either P22121 or P212121.
I did change the number of grid points to 13 on my last integration run.
Distance seems to fluctuate from 303.7 to 298.7 - only 303 at the very beginning and then it drops down to 299 for the rest of the images.

At this point I'm mostly wanting to get a handle on what to do next time I'm collecting data - whether I need to collect finer slices or try and position the crystal in the loop at a different angle, or what.

Thanks for the help,

Jason.

--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds StCELL
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 84155
fx:  +64 9 3737414

On 17/07/2012, at 3:44 PM, Bosch, Juergen wrote:

grep SPOT_RANGE IDXREF.LP should provide you information about that. No idea what the default would be.
How about pointless ?

Something else which might buy you a bit of signal is 

NUMBER_OF_PROFILE_GRID_POINTS_ALONG_ALPHA/BETA=13
NUMBER_OF_PROFILE_GRID_POINTS_ALONG_GAMMA=13

The default for both is 9, you'll end up with a finer profile 13x13x13.
If you grep for DISTANCE in INTEGRATE.LP is it stable ? If not you might want to define which values to refine in the integration step via INTEGRATE(REFINE)=CELL etc.

Jürgen

On Jul 16, 2012, at 11:28 PM, Jason Busby wrote:

Hi,

The autoindexing picks this unit cell pretty much unambiguously, and the profiles look reasonable.  These are crystals of a very large heterodimer (2177 residues), and this unit cell would have 2 heterodimers and 56% solvent, which seems reasonable.  Scaling and merging produce reasonable statistics (I used aimless, not XSCALE):
                                           Overall  InnerShell  OuterShell
Low resolution limit                       19.91     19.91      3.04
High resolution limit                       2.99     16.38      2.99

Rmerge  (within I+/I-)                     0.339     0.040     0.907
Rmerge  (all I+ and I-)                    0.348     0.045     0.949
Rmeas (within I+/I-)                       0.360     0.042     0.994
Rmeas (all I+ & I-)                        0.359     0.046     0.997
Rpim (within I+/I-)                        0.119     0.014     0.393
Rpim (all I+ & I-)                         0.085     0.012     0.291
Rmerge in top intensity bin                0.053        -         - 
Total number of observations             1981569      5075     44784
Total number unique                       112524       338      4559
Mean((I)/sd(I))                             10.6      53.4       2.6
Mn(I) half-set correlation CC(1/2)         0.993     0.999     0.527
Completeness                                98.8      43.7      82.1
Multiplicity                                17.6      15.0       9.8


Rmerge is high in the outer shell, but looks ok to me across the rest of the data.  The oscillation angle is correct.

The native data set also indexes with the same spacegroup and a slightly smaller unit cell (a=134 b=148 c=274), I attributed the difference to the Pt soak.  

The only ambiguity is one of the screw axes, so it may be P22121 or P212121.  My XDS.INP has SPOT_RANGE commented out so I believe the default is to use all the data for indexing.

Cheers,

Jason.
--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 84155
fx:  +64 9 3737414

On 17/07/2012, at 3:02 PM, Bosch, Juergen wrote:

Hi Jason,

if you look at the generated profiles in INTEGRATE.LP do they seem reasonable ?
Does XSCALE.LP produce reasonable I/SigI statistics and expected Rvalues ? If not this might be another hint at wrong cell/spacegroup perhaps.
You can try collecting spots from your whole data set with SPOT_RANGE= [start frame] [end frame] and then index the data. If you get too many strong spots you can select the top 5000 from SPOT.XDS.
Is the oscillation correct in your script ?

Jürgen

P.S. we just collected some data on a 460Å cell

On Jul 16, 2012, at 5:52 PM, Jason Busby wrote:

Hi,

I have a crystal with a large unit cell in P21221 (a=134 b=151 c=276) and I'm wondering if I have a problem with overlaps.  I have a native dataset, and am trying to get phases.  I've collected a Pt soak data set on our home source with a 0.5˚ oscillation angle, but the anomalous signal drops off after about 8Å.  I am wondering if this is a problem due to overlaps at higher resolution.

The Pt dataset has been integrated with XDS, and there don't seem to be too many rejects, but looking at FRAME.CBF it looks like the predicted spots are overlapping at higher resolution.  You can see a zoomed-in part of FRAME.CBF here:
http://imgur.com/1WShV

Should I be concerned with this?  The crystal mosaicity from XDS is 0.25, so fairly low.  What can I do about this, should I try smaller oscillation angles?

Thanks,

Jason.

--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 84155
fx:  +64 9 3737414


......................
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






......................
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






......................
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