Re: [ccp4bb] tNCS incompatible with cell dimensions

[Kevin Jude]

Thanks everybody for your replies. I am having another look at my data in
P1 and will post an update and summary to the list.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431


On Fri, May 31, 2019 at 1:09 PM Kevin Jude  wrote:

> Hello community, I wonder if I could solicit advice about a problematic
> dataset. I plan to solve the structure by molecular replacement and expect
> that the protein is relatively compact, ie not elongated. SAXS data
> supports this expectation.
>
> The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2
> with a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with
> 40% solvent. The native Patterson shows a large peak (12 sigma) suggesting
> a tNCS vector of {0.5, 0.5, 0}.
>
> If you're sharper than me, you may have already spotted the problem - c is
> the long axis of the unit cell, but tNCS constrains the proteins to a plane
> parallel to the a,b plane. Indeed, molecular replacement attempts using
> Phaser will not give a solution in any orthorhombic space group unless I
> turn off packing, and then I get large overlaps in the a,b plane and huge
> gaps along c.
>
> Since I believe that my model is good (or at least the correct shape,
> based on SAXS), I wonder if I'm misinterpreting my crystallographic data.
> Any insights into how to approach this problem would be much appreciated.
>
> --
> Kevin Jude, PhD
> Structural Biology Research Specialist, Garcia Lab
> Howard Hughes Medical Institute
> Stanford University School of Medicine
> Beckman B177, 279 Campus Drive, Stanford CA 94305
> Phone: (650) 723-6431
>
>



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[ccp4bb] AW: [EXTERNAL] [ccp4bb] tNCS incompatible with cell dimensions

[Herman . Schreuder]

Dear Kevin,

It could also be that you have a particular nasty combination of tNCS and 
twinning. Given your packing problems in the ab plane, this would mean that 
your 2-fold parallel to c is generated by twinning and that probably one of the 
21 axes is generated by twinning as well.

With some very clever thinking, you might be able to figure out what the 
appropriate lower-symmetry space group would be, but I would follow the advice 
of John Helliwell: reprocess the data in P1 and run molecular replacement in 
P1. MR is usually quite insensitive to twinning and will produce two solutions 
with equal probability. Once you have the packing, you can try to figure out 
how to best describe this packing in terms of a space group with tNCS. If your 
data collection statistics do not suggest any twinning, you may have some other 
pathology like statistical disorder.

In any case, with this space group problem, you have great opportunity to learn 
a lot about crystallography!

Best,
Herman




Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Kevin 
Jude
Gesendet: Freitag, 31. Mai 2019 22:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] tNCS incompatible with cell dimensions


EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk

Hello community, I wonder if I could solicit advice about a problematic 
dataset. I plan to solve the structure by molecular replacement and expect that 
the protein is relatively compact, ie not elongated. SAXS data supports this 
expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a 
= 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
vector of {0.5, 0.5, 0}.

If you're sharper than me, you may have already spotted the problem - c is the 
long axis of the unit cell, but tNCS constrains the proteins to a plane 
parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser 
will not give a solution in any orthorhombic space group unless I turn off 
packing, and then I get large overlaps in the a,b plane and huge gaps along c.

Since I believe that my model is good (or at least the correct shape, based on 
SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights 
into how to approach this problem would be much appreciated.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431




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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Eleanor Dodson]

Hmm - that sym op means you  have a near C centred cell with spacegroup C 2
2 2 ?

Maybe some of your protein has been chewed up? That does happen?

How good is the diffraction?

Eleanor

On Sat, 1 Jun 2019 at 17:44, Jonathan Cooper <
0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:

> Does the SAXS model contain more than one subunit? If so, I would be
> tempted to go back to the model and try each one separately. This may not
> apply, but if there are monomers in the SAXS model that are related by
> space group symmetry in the crystal, I think the MR would never work. Good
> luck with it! Bests, Jon. Cooper
>
> Sent from Yahoo Mail on Android
> 
>
> On Sat, 1 Jun 2019 at 9:45, Jrh Gmail
>  wrote:
> Dear Kevin
> You could try reindexing into P1, then run Phaser and with its solution as
> input to Zanuda determine the space group.
> Best wishes,
> John
>
> Emeritus Professor of Chemistry John R Helliwell DSc_Physics
>
>
>
>
> On 31 May 2019, at 21:09, Kevin Jude  wrote:
>
> Hello community, I wonder if I could solicit advice about a problematic
> dataset. I plan to solve the structure by molecular replacement and expect
> that the protein is relatively compact, ie not elongated. SAXS data
> supports this expectation.
>
> The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2
> with a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with
> 40% solvent. The native Patterson shows a large peak (12 sigma) suggesting
> a tNCS vector of {0.5, 0.5, 0}.
>
> If you're sharper than me, you may have already spotted the problem - c is
> the long axis of the unit cell, but tNCS constrains the proteins to a plane
> parallel to the a,b plane. Indeed, molecular replacement attempts using
> Phaser will not give a solution in any orthorhombic space group unless I
> turn off packing, and then I get large overlaps in the a,b plane and huge
> gaps along c.
>
> Since I believe that my model is good (or at least the correct shape,
> based on SAXS), I wonder if I'm misinterpreting my crystallographic data.
> Any insights into how to approach this problem would be much appreciated.
>
> --
> Kevin Jude, PhD
> Structural Biology Research Specialist, Garcia Lab
> Howard Hughes Medical Institute
> Stanford University School of Medicine
> Beckman B177, 279 Campus Drive, Stanford CA 94305
> Phone: (650) 723-6431
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> --
>
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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Jonathan Cooper]

Does the SAXS model contain more than one subunit? If so, I would be tempted to 
go back to the model and try each one separately. This may not apply, but if 
there are monomers in the SAXS model that are related by space group symmetry 
in the crystal, I think the MR would never work. Good luck with it! Bests, Jon. 
Cooper

Sent from Yahoo Mail on Android 
 
  On Sat, 1 Jun 2019 at 9:45, Jrh Gmail wrote:   Dear 
KevinYou could try reindexing into P1, then run Phaser and with its solution as 
input to Zanuda determine the space group. Best wishes,John 

Emeritus Professor of Chemistry John R Helliwell DSc_Physics 



On 31 May 2019, at 21:09, Kevin Jude  wrote:


Hello community, I wonder if I could solicit advice about a problematic 
dataset. I plan to solve the structure by molecular replacement and expect that 
the protein is relatively compact, ie not elongated. SAXS data supports this 
expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a 
= 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
vector of {0.5, 0.5, 0}.
If you're sharper than me, you may have already spotted the problem - c is the 
long axis of the unit cell, but tNCS constrains the proteins to a plane 
parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser 
will not give a solution in any orthorhombic space group unless I turn off 
packing, and then I get large overlaps in the a,b plane and huge gaps along c.
Since I believe that my model is good (or at least the correct shape, based on 
SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights 
into how to approach this problem would be much appreciated.
--
Kevin Jude, PhDStructural Biology Research Specialist, Garcia LabHoward Hughes 
Medical InstituteStanford University School of MedicineBeckman B177, 279 Campus 
Drive, Stanford CA 94305Phone: (650) 723-6431


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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Jrh Gmail]

Dear Kevin
You could try reindexing into P1, then run Phaser and with its solution as 
input to Zanuda determine the space group. 
Best wishes,
John 

Emeritus Professor of Chemistry John R Helliwell DSc_Physics 




> On 31 May 2019, at 21:09, Kevin Jude  wrote:
> 
> Hello community, I wonder if I could solicit advice about a problematic 
> dataset. I plan to solve the structure by molecular replacement and expect 
> that the protein is relatively compact, ie not elongated. SAXS data supports 
> this expectation.
> 
> The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with 
> a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
> solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
> vector of {0.5, 0.5, 0}.
> 
> If you're sharper than me, you may have already spotted the problem - c is 
> the long axis of the unit cell, but tNCS constrains the proteins to a plane 
> parallel to the a,b plane. Indeed, molecular replacement attempts using 
> Phaser will not give a solution in any orthorhombic space group unless I turn 
> off packing, and then I get large overlaps in the a,b plane and huge gaps 
> along c.
> 
> Since I believe that my model is good (or at least the correct shape, based 
> on SAXS), I wonder if I'm misinterpreting my crystallographic data. Any 
> insights into how to approach this problem would be much appreciated.
> 
> --
> Kevin Jude, PhD
> Structural Biology Research Specialist, Garcia Lab
> Howard Hughes Medical Institute
> Stanford University School of Medicine
> Beckman B177, 279 Campus Drive, Stanford CA 94305
> Phone: (650) 723-6431
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Kevin Jude]

Thanks Diana - Indexing on strong reflections (STRONG_PIXEL=50 in xds) does
identify C222 as a possibility with the same dimensions as the P222 cell.
This doesn't solve my problem, though, since the centering operation just
replaces the tNCS and doesn't relieve the crowding.

Best wishes
Kevin
--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431


On Fri, May 31, 2019 at 1:35 PM Diana Tomchick <
diana.tomch...@utsouthwestern.edu> wrote:

> Your native Patterson indicates pseudo C-centering. Are you sure you don’t
> have space group C222(1)?
>
> If your space group is correct, it’s still pseudo C-centered. You should
> see that in the intensity-weighted reciprocal lattice.
>
> You could try re-indexing on just the most intense spots to give you a
> data set indexed in a C-centered lattice. Use that data to solve via MR,
> then convert to the data indexed in the actual space group.
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On May 31, 2019, at 3:09 PM, Kevin Jude  wrote:
>
> Hello community, I wonder if I could solicit advice about a problematic
> dataset. I plan to solve the structure by molecular replacement and expect
> that the protein is relatively compact, ie not elongated. SAXS data
> supports this expectation.
>
> The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2
> with a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with
> 40% solvent. The native Patterson shows a large peak (12 sigma) suggesting
> a tNCS vector of {0.5, 0.5, 0}.
>
> If you're sharper than me, you may have already spotted the problem - c is
> the long axis of the unit cell, but tNCS constrains the proteins to a plane
> parallel to the a,b plane. Indeed, molecular replacement attempts using
> Phaser will not give a solution in any orthorhombic space group unless I
> turn off packing, and then I get large overlaps in the a,b plane and huge
> gaps along c.
>
> Since I believe that my model is good (or at least the correct shape,
> based on SAXS), I wonder if I'm misinterpreting my crystallographic data.
> Any insights into how to approach this problem would be much appreciated.
>
> --
> Kevin Jude, PhD
> Structural Biology Research Specialist, Garcia Lab
> Howard Hughes Medical Institute
> Stanford University School of Medicine
> Beckman B177, 279 Campus Drive, Stanford CA 94305
> Phone: (650) 723-6431
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
>
> UT Southwestern
>
> Medical Center
>
> The future of medicine, today.
>



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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Boaz Shaanan]

Wouldn't c222 or c222(1) be included in the Phaser run if all orthorhombic sg's were requested?
Boaz

Boaz Shaanan, Ph.D.
Department of Life Sciences
Ben Gurion University of the Negev
Beer Sheva
Israel



On May 31, 2019 23:37, Diana Tomchick  wrote:


Your native Patterson indicates pseudo C-centering. Are you sure you don’t have space group C222(1)?


If your space group is correct, it’s still pseudo C-centered. You should see that in the intensity-weighted reciprocal lattice.


You could try re-indexing on just the most intense spots to give you a data set indexed in a C-centered lattice. Use that data to solve via MR, then convert to the data indexed in the actual space group.


Diana



**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)


On May 31, 2019, at 3:09 PM, Kevin Jude  wrote:



Hello community, I wonder if I could solicit advice about a problematic dataset. I plan to solve the structure by molecular replacement and expect that the protein is relatively compact, ie not elongated. SAXS data supports this expectation.



The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS vector of {0.5,
 0.5, 0}.


If you're sharper than me, you may have already spotted the problem - c is the long axis of the unit cell, but tNCS constrains the proteins to a plane parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser will not give
 a solution in any orthorhombic space group unless I turn off packing, and then I get large overlaps in the a,b plane and huge gaps along c.


Since I believe that my model is good (or at least the correct shape, based on SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights into how to approach this problem would be much appreciated.












--

Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute

Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: 
(650) 723-6431















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UT
 Southwestern 




Medical Center







The future of medicine, today.






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Re: [ccp4bb] tNCS incompatible with cell dimensions

[Diana Tomchick]

Your native Patterson indicates pseudo C-centering. Are you sure you don’t have 
space group C222(1)?

If your space group is correct, it’s still pseudo C-centered. You should see 
that in the intensity-weighted reciprocal lattice.

You could try re-indexing on just the most intense spots to give you a data set 
indexed in a C-centered lattice. Use that data to solve via MR, then convert to 
the data indexed in the actual space group.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On May 31, 2019, at 3:09 PM, Kevin Jude 
mailto:kj...@stanford.edu>> wrote:

Hello community, I wonder if I could solicit advice about a problematic 
dataset. I plan to solve the structure by molecular replacement and expect that 
the protein is relatively compact, ie not elongated. SAXS data supports this 
expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2 with a 
= 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with 40% 
solvent. The native Patterson shows a large peak (12 sigma) suggesting a tNCS 
vector of {0.5, 0.5, 0}.

If you're sharper than me, you may have already spotted the problem - c is the 
long axis of the unit cell, but tNCS constrains the proteins to a plane 
parallel to the a,b plane. Indeed, molecular replacement attempts using Phaser 
will not give a solution in any orthorhombic space group unless I turn off 
packing, and then I get large overlaps in the a,b plane and huge gaps along c.

Since I believe that my model is good (or at least the correct shape, based on 
SAXS), I wonder if I'm misinterpreting my crystallographic data. Any insights 
into how to approach this problem would be much appreciated.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431




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UT Southwestern


Medical Center



The future of medicine, today.




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[ccp4bb] tNCS incompatible with cell dimensions

[Kevin Jude]

Hello community, I wonder if I could solicit advice about a problematic
dataset. I plan to solve the structure by molecular replacement and expect
that the protein is relatively compact, ie not elongated. SAXS data
supports this expectation.

The crystals diffract to 2.6 Å resolution and appear to be in P 21 21 2
with a = 49, b = 67, c = 94, which should fit <=2 molecules in the ASU with
40% solvent. The native Patterson shows a large peak (12 sigma) suggesting
a tNCS vector of {0.5, 0.5, 0}.

If you're sharper than me, you may have already spotted the problem - c is
the long axis of the unit cell, but tNCS constrains the proteins to a plane
parallel to the a,b plane. Indeed, molecular replacement attempts using
Phaser will not give a solution in any orthorhombic space group unless I
turn off packing, and then I get large overlaps in the a,b plane and huge
gaps along c.

Since I believe that my model is good (or at least the correct shape, based
on SAXS), I wonder if I'm misinterpreting my crystallographic data. Any
insights into how to approach this problem would be much appreciated.

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431



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