Difficult dataset and refinement--P422? I422?Twinning?

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Zhou, Tongqing (NIH/VRC) [E]

7 May 2012 7 May '12

2:50 p.m.

Dear All, I have collected several dataset for an antibody:antigen complex. It apparently indexed to P4 space group with large cell dimensions of 191.7000/191.7000/311.0540. When I processed as P1 and ran Pointless, it suggested P422 as space group. P4212 gives the best MR solution with 6 mol/ASU (18 chains total ). However, ridigbody and subsequent refinement with TLS could not reduce the r and r-free which stayed at ~48%. Tried to go P4, P222...(12 mol/ASU) with twinning, but the refinement failed due to insufficient computer memory (12 GB), it was also impossible to refine in P1 due to the same reason. But for some data sets, HKL2000 also gave I4 space group with c axis reduced to 1/3 of that of P4. With the I4 data (~2.7 A resolution), I422 was the best MR space group (1 mol/ASU), the refinement went down to r=29% and r-free=34%, but won't go down further. Please advise if it's possible to use the data Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4609B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ******************************************************************

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Kelly Daughtry

7 May 7 May

3:46 p.m.

New subject: Difficult dataset and refinement--P422? I422?Twinning?

Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422?

...

From the P422_xtriage.log:

The full list of Patterson peaks is: x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03) ( 0.500, 0.500, 0.187 ) : 4.873 (9.347e-01) If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible: space group operator unit cell of reference setting I 4 2 2 (a,b,3*c) x+1/2, y+1/2, z+1/6 (191.70, 191.70, 103.68, 90.00, 90.00, 90.00) Secondly, for your I4 datasets, if the resoluiton of your I422_xtriage.log file is correct (2.76 angstroms), then an r=29% and r-free=34% is not too far off, using the general rule of thumb that a structure is close to completion when the r-factors are 10 times the resolution. For example, resolution of 2.7 a r-factor around 27% should indicate that the structure is close to finished. Ultimately, it's your manual inspection and judgement (aside from statistics) that dictate whether you think the structure to be fully refined. Hope that helps, Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Mon, May 7, 2012 at 10:50 AM, Zhou, Tongqing (NIH/VRC) [E] wrote:

...

Dear All,

I have collected several dataset for an antibody:antigen complex. It apparently indexed to P4 space group with large cell dimensions of 191.7000/191.7000/311.0540. When I processed as P1 and ran Pointless, it suggested P422 as space group. P4212 gives the best MR solution with 6 mol/ASU (18 chains total ). However, ridigbody and subsequent refinement with TLS could not reduce the r and r-free which stayed at ~48%. Tried to go P4, P222…(12 mol/ASU) with twinning, but the refinement failed due to insufficient computer memory (12 GB), it was also impossible to refine in P1 due to the same reason.

But for some data sets, HKL2000 also gave I4 space group with c axis reduced to 1/3 of that of P4. With the I4 data (~2.7 A resolution), I422 was the best MR space group (1 mol/ASU), the refinement went down to r=29% and r-free=34%, but won’t go down further.

Please advise if it’s possible to use the data

Tongqing

Tongqing Zhou, Ph.D.

Staff Scientist

Structural Biology Section

Vaccine Research Center, NIAID/NIH

Building 40, Room 4609B

40 Convent Drive, MSC3027

Bethesda, MD 20892

(301) 594-8710 (Tel)

(301) 793-0794 (Cell)

(301) 480-2658 (Fax)

******************************************************************************

The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives.

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Nathaniel Echols

4:18 p.m.

New subject: Difficult dataset and refinement--P422? I422?Twinning?

On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry wrote:

...

Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422?

From the P422_xtriage.log:

The full list of Patterson peaks is:

x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03)

One word of caution: an exceptionally high off-origin peak can mean that the unit cell was measured too large, and you've integrated extra reflections that are really non-existent. (I'm not sure what the threshold for this is, but 80% seems pretty large.) Splitting and various indexing artifacts can sometimes lead to this. I'd recommend running labelit.index on the images and seeing what it thinks the lattice should be. -Nat

Kelly Daughtry

4:50 p.m.

New subject: Difficult dataset and refinement--P422? I422?Twinning?

I agree. The fact that two "crystals forms" appear, with related unit cells: P422 data: unit_cell = 191.6999969 191.6999969 311.0539856 90 90 90 space_group = "P 4 2 2" I422 data: unit_cell = 191.783 191.783 103.775 90 90 90 space_group = "I 4 2 2" indicated to me you have mis-indexed the P422 data. It is likely I422. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Mon, May 7, 2012 at 12:18 PM, Nathaniel Echols wrote:

...

On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry wrote:

...
Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422?

From the P422_xtriage.log:

The full list of Patterson peaks is:

x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03)

One word of caution: an exceptionally high off-origin peak can mean that the unit cell was measured too large, and you've integrated extra reflections that are really non-existent. (I'm not sure what the threshold for this is, but 80% seems pretty large.) Splitting and various indexing artifacts can sometimes lead to this. I'd recommend running labelit.index on the images and seeing what it thinks the lattice should be.

-Nat _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Zhou, Tongqing (NIH/VRC) [E]

5:08 p.m.

New subject: Difficult dataset and refinement--P422? I422?Twinning?

Hi Kelly and Nat, Thanks for your quick reply. The two space groups come out depending on which frames I started to index. Actually, in the P422 xtriage outpout, it stated it could be I422 with C'=C/3: ____________________________________________ The full list of Patterson peaks is: x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03) ( 0.500, 0.500, 0.187 ) : 4.873 (9.347e-01) If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible: space group operator unit cell of reference setting I 4 2 2 (a,b,3*c) x+1/2, y+1/2, z+1/6 (191.70, 191.70, 103.68, 90.00, 90.00, 90.00) ____________________________________________________________________ When I indexed as I422, some of the spots were not picked, but scaled data went to 2.65 A; P422 could cover all the spots, but resolution could only reach ~2.9A, however, I did see 5 sigma spots around 2.6/2.7. This might indicate I have a "perfectly" split crystal so that those spots looked like they belong to a P4 space group were actually from other crystals. Regards, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4609B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ****************************************************************** -----Original Message----- From: Kelly Daughtry [mailto:[email protected]] Sent: Monday, May 07, 2012 12:50 PM To: PHENIX user mailing list Subject: Re: [phenixbb] Difficult dataset and refinement--P422? I422?Twinning? I agree. The fact that two "crystals forms" appear, with related unit cells: P422 data: unit_cell = 191.6999969 191.6999969 311.0539856 90 90 90 space_group = "P 4 2 2" I422 data: unit_cell = 191.783 191.783 103.775 90 90 90 space_group = "I 4 2 2" indicated to me you have mis-indexed the P422 data. It is likely I422. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Mon, May 7, 2012 at 12:18 PM, Nathaniel Echols wrote:

...

On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry wrote:

...
Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422?

From the P422_xtriage.log:

The full list of Patterson peaks is:

x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03)

One word of caution: an exceptionally high off-origin peak can mean that the unit cell was measured too large, and you've integrated extra reflections that are really non-existent. (I'm not sure what the threshold for this is, but 80% seems pretty large.) Splitting and various indexing artifacts can sometimes lead to this. I'd recommend running labelit.index on the images and seeing what it thinks the lattice should be.

-Nat _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Bosch, Juergen

5:33 p.m.

New subject: Difficult dataset and refinement--P422? I422?Twinning?

Why don't you index over the whole dataset (XDS) ? Or at least over a couple of images (Mosflm) Jürgen On May 7, 2012, at 12:50 PM, Kelly Daughtry wrote: I agree. The fact that two "crystals forms" appear, with related unit cells: P422 data: unit_cell = 191.6999969 191.6999969 311.0539856 90 90 90 space_group = "P 4 2 2" I422 data: unit_cell = 191.783 191.783 103.775 90 90 90 space_group = "I 4 2 2" indicated to me you have mis-indexed the P422 data. It is likely I422. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Mon, May 7, 2012 at 12:18 PM, Nathaniel Echols mailto:[email protected]> wrote: On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry mailto:[email protected]> wrote: Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422?

...

From the P422_xtriage.log:

The full list of Patterson peaks is: x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03) One word of caution: an exceptionally high off-origin peak can mean that the unit cell was measured too large, and you've integrated extra reflections that are really non-existent. (I'm not sure what the threshold for this is, but 80% seems pretty large.) Splitting and various indexing artifacts can sometimes lead to this. I'd recommend running labelit.index on the images and seeing what it thinks the lattice should be. -Nat _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb ...................... 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://web.mac.com/bosch_lab/

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Bosch, Juergen
Kelly Daughtry
Nathaniel Echols
Zhou, Tongqing (NIH/VRC) [E]