[ccp4bb] Coot Find Ligand Places Ligand In Density Outside Where Model Is Built

[Roger Shek]

I am new to this, so forgive the perhaps naive question. Does anyone know
why Coot is placing ligand outside the electron density where my model is
built. I have put the cursor to where I want it to search, but when it
fits, it places the ligand in the equivalent spot, but outside the model
and not where the cursor was? I tried renaming the chains, but now it the
find ligand doesn't even find anything even though there is clear density
there. Thanks in advance.

Roger

-- 
Roger Shek

Stony Brook University
Graduate Student in Biochemistry and Structural Biology (PhD)
Stony Brook Integrative Structural Biology Organization

Cell: (808) 386-3879

Re: [ccp4bb] CCP4BB Digest - 10 Apr 2017 to 11 Apr 2017 (#2017-100)

[Jim Pflugrath]

Let's step back a little bit first:

Do these 3 to 4 diffraction data sets (when kept separate) process nicely
individually and have good Rmeas and other results when scaled separately
from each other?  If so, then do they also have the same unit cell
dimensions?

Jim


 Date:Tue, 11 Apr 2017 10:46:25 +0800

> From:高艺娜 
> Subject: Some problems in data processing
>
> Dear all,
> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000
> program to meet requirements for data completeness, but the problem is the
> Rmerge value was too high to go on every time.
> Did anyone have met the same problem and how to solve it? or some tips for
> solve this problem？
>
> All comments will be appreciated!
>
>

[ccp4bb] Glycoprotein expression question

[Bernhard Rupp]

Hi Fellows,

 

a humble question for our glyco-expressionists: 

 

I have mutated out the Asns of the N-glycoslation consensus sites for Asp 

(Asp simply because the PNGaseF treated protein stays stable so I thought
that might be a good guess) 

and indeed the unglycosilated mutant expresses well and gets secreted as
planned. 

 

But rumor has it that glycoproteins that are mutated to non-glyc often are
not processed correctly and 

that we had just dumb luck. 

 

May I poll the educated opinion of the erudite here?

 

Cheers, BR

 

--

Bernhard Rupp

Crystallographiae Vindicis Militum Ordo

  http://www.hofkristallamt.org/

  b...@hofkristallamt.org

+1 925 209 7429

+43 767 571 0536

--

:(){ :|: & };:

--

 

[ccp4bb] High Rfree: Phasing issue or partial crystal disorder

[Pravinkumar Jagtap]

Dear All,
I am stuck with this problem for 2 months and hope you could help.

We have a 2.1 A dataset for a 380 amino acid long protein. The space group
is I4 (single molecule in asymmetric unit, 48% solvent content) and the
dataset is quite perfect (no obvious pathologies). The protein itself is
organised in 2 lobes (N and C terminal lobes). The sequence identity to
nearest homologue structure is 17%.

We could  get the phases by SeMet SAD phasing (3A resolution dataset, 5
SeMet (excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe
and 2 partial occupancy (~0.5 each; present on surface) SeMet in N-terminal
lobe). Automated  model building (at 2.1 A) yielded nice model for the
C-terminal lobe (215 residues)  and manually I could build parts (around 80
residues) of N-terminal lobe with high confidence. In addition we could
also build a ligand which is sandwiched between C and N terminal lobe.

However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some
patchy density left at the N terminal lobe but as it is discontinuous, I
cannot build anything in it (except lots of water molecules). In total I am
missing around 85 residues. These residues are predicted to be present in
secondary structure (and not flexible).

As I have around 75-80% model built, I would expect that I would have all
the phases and  should get nice density for the remaining part. But as I
dont see it, could the rest part be flexible? But again, this is not
reflected in the R factors (I would then expect low Rfree).

Could it be that I still lack phases (due to partial occupancy of SeMeth in
N-terminal lobe ) and have to try to get them by heavy metal soaking, or
there is disorder in the N-terminal lobe? I have also tried solving
different datasets for same crystal but this has not been useful.

Regards,
Pravin.

Re: [ccp4bb] Some problems in data processing

[Kevin Jude]

You don't say what you mean by "too high", but if you are in a space group
with multiple possible indexing conventions (eg different choices for the
direction of b in a polar spacegroup) you can get very high Rmerge values.
In the scalepack section of the old HKL manual (
http://hkl-xray.com/hkl-manual) there is a Reindexing scenario that
explains how to approach this problem; the HKL matrices can be used as
macros in HKL2000.

Best wishes,
kmj

On Mon, Apr 10, 2017 at 7:46 PM, 高艺娜  wrote:

> Dear all,
> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000
> program to meet requirements for data completeness, but the problem is the
> Rmerge value was too high to go on every time.
> Did anyone have met the same problem and how to solve it? or some tips for
> solve this problem？
>
> All comments will be appreciated!
>
> Best Regards,
>
>


-- 
Kevin Jude, PhD
Research Specialist, Garcia Lab
Departments of Molecular & Cellular Physiology and Structural Biology
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431

Re: [ccp4bb] Some problems in data processing

[Harry Powell]

Hi

First thing I would do is throw xia2 at it (from ccp4i2, of course ;-)) and go 
and have a cup of coffee, and see if the statistics were similar after a Danish 
pastry.

If the stats are better from xia2 than from HKL, go with the xia2 processing, 
if they are the same (or worse) I'd look more closely at Mark van Raaij's 
answer (added below) - my _initial_ assumption in this case would be that the 
crystals aren't isomorphous and the data can't be merged successfully, but it's 
hard to say much without more information than you've given us.

Back to your original processing 

- What are the stats like for each individual crystal? 
- Do any of the different datasets merge together with good statistics? 
- Is the crystal system one where you can have alternative indexing (e.g. 
hexagonal, tetragonal)? - Pointless should be your friend here

Of course, something I  would look at would be the various discussions here and 
elsewhere on the usefulness of Rmerge as an indicator of data quality (hint: it 
isn't very good compared to other measures).

On 11 Apr 2017, at 10:23, Frank von Delft wrote:

> Quite a few papers on this in recent years, including from the Sasha Popov 
> (ESRF) and Wayne Hendrickson - don't have references to hand, the Google 
> should sort you out.
> 
> And then of course the CCP4 program BLEND - that's both published and 
> runnable.
> 
> But overall:  this is the top problem in data processing at the moment, 
> keeping a lot of smart people very busy.  If that makes you feel better ;)  
> Maybe they'll comment.
> 
> 
> On 11/04/2017 03:46, 高艺娜 wrote:
>> Dear all,
>> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000 
>> program to meet requirements for data completeness, but the problem is the 
>> Rmerge value was too high to go on every time.
>> Did anyone have met the same problem and how to solve it? or some tips for 
>> solve this problem？
>> 
>> All comments will be appreciated!
>> 
>> Best Regards,
>> 

> Hi Gaoyina,
> 
> I guess your crystals ended up in slightly different lattices or with 
> slightly different ordering during flash-cooling. 
> I this case can see two possible solutions:
> - collect more complete data from a single crystal, you may have to 
> "sacrifice" some resolution by collecting shorter images or attenuating the 
> beam somewhat.
> - try to flash-cool the crystals in a more reproducible way, so they are more 
> similar to each other.
> Another possibility might be that you have too many spot overlaps, in this 
> case you may need to collect thinner-sliced images, offset the detector, put 
> the detector further away (i.e. lower resolution), or measure a crystal with 
> a different orientation (try put the spindle rotation more or less around the 
> long cell axis).
> In any of these cases I think you will need to collect more data.
> With the current data, in Mosflm data processing you may be able to get more 
> complete data with the SEPARATION CLOSE option, if you did not invoke this 
> already. In other data processing programs there surely are similar options. 
> On the other hand, in the automated data processing pipelines that many 
> synchrotrons use now they are probably already activated when necessary (at 
> least I have rarely been able to improve on automatically processed data when 
> doing it myself later...)
> 
> Greetings,
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> http://wwwuser.cnb.csic.es/~mjvanraaij
> 

[ccp4bb] Beamtime @ SLS

[Meitian Wang]

===
SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY AT SLS
===

Proposal application deadline: Thursday, April 20, 2017

Periods:
July 1, 2017 - December 31, 2017 (Normal / Test proposals)
July 1, 2017 - June 30, 2019 (Long-term proposals)

Proposal submission:
http://www.psi.ch/sls/px-beamlines-call-for-proposals 


What's New (http://www.psi.ch/sls/pxi/pxi ) 
- X06SA
Fast beam size changing from 5 x 5 to 80 x 80 micron^2, one-micron beam 
available
EIGER 16M detector (133 Hz)
Continous grid scan (100Hz)
Serial data collection GUI (CY+)
- X06DA
Rapid access mode for experimental phasing, especially native-SAD 
(contact directly vincent.olie...@psi.ch )
- Sample changer
30 second sample exchange time

Beamline characteristics and features
X06SA Beamline (http://www.psi.ch/sls/pxi/pxi )
X06DA Beamline (http://www.psi.ch/sls/pxiii/pxiii 
)
X10SA Beamline (http://www.psi.ch/sls/pxii/pxii 
)

Best regards,

The MX group at SLS

__
Meitian Wang
Swiss Light Source at Paul Scherrer Institut
CH-5232 Villigen PSI - http://www.psi.ch/sls/ 
Phone: +41 56 310 4175

Re: [ccp4bb] Some problems in data processing

[Frank von Delft]

Quite a few papers on this in recent years, including from the Sasha 
Popov (ESRF) and Wayne Hendrickson - don't have references to hand, the 
Google should sort you out.


And then of course the CCP4 program BLEND - that's both published and 
runnable.


But overall:  this is the top problem in data processing at the moment, 
keeping a lot of smart people very busy.  If that makes you feel better 
;)  Maybe they'll comment.



On 11/04/2017 03:46, 高艺娜 wrote:

Dear all,
For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000 
program to meet requirements for data completeness, but the problem is the 
Rmerge value was too high to go on every time.
Did anyone have met the same problem and how to solve it? or some tips for 
solve this problem？

All comments will be appreciated!

Best Regards,

Re: [ccp4bb] off topic: MacPymol and macOS Sierra

[Freia von Raussendorf]

Hi,

I had this problem with Yosemite. Clicking on the icon still doesn’t open the 
program, but I can start it using the sh command in XQuartz..
I’m not sure if there was something else I had to do in order for this to work 
(it’s been a while since I started using it like this), but perhaps just try it.

Cheers,
Freia


> On 11 Apr 2017, at 00:57, Peter Hsu  wrote:
> 
> Hi all, 
> 
> Sorry for the very off topic message, but I recently upgraded my OS to Sierra 
> and all of a sudden MacPymol has stopped working for me. Clicking on the 
> program just gives the appearance of it about to start by showing the icon on 
> the dock, and then just blinks out, without ever starting. My xcode and 
> xquartz is up to date, so I'm not sure where the problem is. Any help is much 
> appreciated!
> 
> Thanks,
> Peter

[ccp4bb] off topic: Change of bottom filter

[Adriana Sene]

Dear All
I am wondering if its possible to change the bottom filter of the sepharose
12 gel filteration column.

 best
Adriana

Re: [ccp4bb] Some problems in data processing

[Mark J van Raaij]

Hi Gaoyina,

I guess your crystals ended up in slightly different lattices or with slightly 
different ordering during flash-cooling. 
I this case can see two possible solutions:
- collect more complete data from a single crystal, you may have to "sacrifice" 
some resolution by collecting shorter images or attenuating the beam somewhat.
- try to flash-cool the crystals in a more reproducible way, so they are more 
similar to each other.
Another possibility might be that you have too many spot overlaps, in this case 
you may need to collect thinner-sliced images, offset the detector, put the 
detector further away (i.e. lower resolution), or measure a crystal with a 
different orientation (try put the spindle rotation more or less around the 
long cell axis).
In any of these cases I think you will need to collect more data.
With the current data, in Mosflm data processing you may be able to get more 
complete data with the SEPARATION CLOSE option, if you did not invoke this 
already. In other data processing programs there surely are similar options. On 
the other hand, in the automated data processing pipelines that many 
synchrotrons use now they are probably already activated when necessary (at 
least I have rarely been able to improve on automatically processed data when 
doing it myself later...)

Greetings,

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

> On 11 Apr 2017, at 04:46, 高艺娜  wrote:
> 
> Dear all,
> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000 
> program to meet requirements for data completeness, but the problem is the 
> Rmerge value was too high to go on every time.
> Did anyone have met the same problem and how to solve it? or some tips for 
> solve this problem？
> 
> All comments will be appreciated!
> 
> Best Regards,
>