Hi Tongqin, I second Dorothee's point that we need more information to resolve this problem. If you still have this problem please send me inputs files (data, model, .eff from last refinement, as well as ligand .CIF files if any) and I will look into this once I have files. Pavel On 8/31/16 09:29, Dorothee Liebschner wrote:

Hi Tongqin,

It is a bit difficult to diagnose with the information you provided. Could you please answer the following questions?

- Did you also try to reset the B-factors to similar values than neighboring atoms? F.ex. the average B-factor in the model could be 50 A**2, but in the ligand region, it could be lower, let's say 20 A**2. Then the starting value using average B is quite far from the likely B-factor of the ligand.

- What refinement strategy do you apply? How many macro-cycles? Do you use any non-default parameters for B-factor refinement?

- Which Phenix version are you using?

Best wishes,

Dorothee

On Wed, Aug 31, 2016 at 7:12 AM, Zhou, Tongqing (NIH/VRC) [E] mailto:[email protected]> wrote:

Dear All,

I am refining a structure with diffraction to 2.2A resolution and 95 % overall completeness. Now the Rs are at 18% and 23%, respectively. But the program failed to refine B factors for several ligands, atoms had high B factors while showing positive Fo-Fc map around them. Occupancy was set to 1 and I also tried to reset B to mean B of the whole PDB. Any of you seeing this phenomenon? Thanks!

Shown below is HEPES that has high B factor and positive density around it after refinement:

Hi Christian, Could you answer the following questions: - what is the resolution of the data set? - what is average B of the protein? - what are the starting B values for the ligand? - did you try lowering starting B for the ligand and repeat the refinement? Is this behavior reproducible with different refinement options? Best wishes, Dorothee On Sun, Oct 30, 2016 at 11:06 AM, Christian Roth

wrote:

Hi Phenix Team,

regarding that ligand issue in this thread, we observed the same with a ligand in one of our structures now. After refinement the ligand is surrounded by pos diff density and the B-Factors are around 200 (way above any useful range in that case).

The cif was generated using elbow and phenix runs with options Real space refinement, TLS on, individual B, and optimizing weights. Nothing crazy.

Could you find a solution for the previous case and give us some tips?

Cheers

Christian

Am 06.09.2016 um 01:44 schrieb Pavel Afonine:

Hi Tongqin,

I second Dorothee's point that we need more information to resolve this problem. If you still have this problem please send me inputs files (data, model, .eff from last refinement, as well as ligand .CIF files if any) and I will look into this once I have files.

Pavel

On 8/31/16 09:29, Dorothee Liebschner wrote:

Hi Tongqin,

It is a bit difficult to diagnose with the information you provided. Could you please answer the following questions?

- Did you also try to reset the B-factors to similar values than neighboring atoms? F.ex. the average B-factor in the model could be 50 A**2, but in the ligand region, it could be lower, let's say 20 A**2. Then the starting value using average B is quite far from the likely B-factor of the ligand.

- What refinement strategy do you apply? How many macro-cycles? Do you use any non-default parameters for B-factor refinement?

- Which Phenix version are you using?

Best wishes,

Dorothee

On Wed, Aug 31, 2016 at 7:12 AM, Zhou, Tongqing (NIH/VRC) [E] < [email protected]> wrote:

...

Dear All,

I am refining a structure with diffraction to 2.2A resolution and 95 % overall completeness. Now the Rs are at 18% and 23%, respectively. But the program failed to refine B factors for several ligands, atoms had high B factors while showing positive Fo-Fc map around them. Occupancy was set to 1 and I also tried to reset B to mean B of the whole PDB. Any of you seeing this phenomenon? Thanks!

Shown below is HEPES that has high B factor and positive density around it after refinement:

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

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

Hi Christian, OK, it looks you found a way to prevent the positive density by using "realistic" starting B values (of the order of that of surrounding protein atoms). Did you also try to increase the number of macro-cycles? Best wishes, Dorothee On Mon, Oct 31, 2016 at 9:11 AM, Christian Roth wrote:

Hi Dorothee

to answer your questions

The resolution is about 2.0 Ang and the average B around 55. The Bfactor from the ligand was set to the default of 20 by Coot when placing the ligands.

It seems that setting the B-value to the average B of the structure nearly solves the problem (most of the ligand fine, just parts with unusuallly high values). Setting it to a bit higher values 70 for a start and the ligand refines fine. I noticed that in the first macrocycle with and initial factor of 20, the B's are nearly unchanged (20 +- 2) Red diff density appears as expected. In the second cycle The minimizer let the B-factors "explode" to about 200 and that basically is the overkill and it never recovers.

HTH

Cheers

Christian

Am 31.10.2016 um 15:59 schrieb Dorothee Liebschner:

Hi Christian,

Could you answer the following questions:

- what is the resolution of the data set? - what is average B of the protein? - what are the starting B values for the ligand? - did you try lowering starting B for the ligand and repeat the refinement? Is this behavior reproducible with different refinement options?

Best wishes,

Dorothee

On Sun, Oct 30, 2016 at 11:06 AM, Christian Roth < [email protected]> wrote:

...

Hi Phenix Team,

regarding that ligand issue in this thread, we observed the same with a ligand in one of our structures now. After refinement the ligand is surrounded by pos diff density and the B-Factors are around 200 (way above any useful range in that case).

The cif was generated using elbow and phenix runs with options Real space refinement, TLS on, individual B, and optimizing weights. Nothing crazy.

Could you find a solution for the previous case and give us some tips?

Cheers

Christian

Am 06.09.2016 um 01:44 schrieb Pavel Afonine:

Hi Tongqin,

I second Dorothee's point that we need more information to resolve this problem. If you still have this problem please send me inputs files (data, model, .eff from last refinement, as well as ligand .CIF files if any) and I will look into this once I have files.

Pavel

On 8/31/16 09:29, Dorothee Liebschner wrote:

Hi Tongqin,

It is a bit difficult to diagnose with the information you provided. Could you please answer the following questions?

- Did you also try to reset the B-factors to similar values than neighboring atoms? F.ex. the average B-factor in the model could be 50 A**2, but in the ligand region, it could be lower, let's say 20 A**2. Then the starting value using average B is quite far from the likely B-factor of the ligand.

- What refinement strategy do you apply? How many macro-cycles? Do you use any non-default parameters for B-factor refinement?

- Which Phenix version are you using?

Best wishes,

Dorothee

On Wed, Aug 31, 2016 at 7:12 AM, Zhou, Tongqing (NIH/VRC) [E] < [email protected]> wrote:

...

Dear All,

I am refining a structure with diffraction to 2.2A resolution and 95 % overall completeness. Now the Rs are at 18% and 23%, respectively. But the program failed to refine B factors for several ligands, atoms had high B factors while showing positive Fo-Fc map around them. Occupancy was set to 1 and I also tried to reset B to mean B of the whole PDB. Any of you seeing this phenomenon? Thanks!

Shown below is HEPES that has high B factor and positive density around it after refinement:

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

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailm an/listinfo/phenixbb Unsubscribe: [email protected]

Hi Christian, somehow this escaped my attention... so was this solved in the end? If not please send me files and I will find a solution. Pavel On 10/31/16 10:55, Christian Roth wrote:

Not more than 5 I'm afraid. I don't know if it would find the right B's using 10 cycles and more, but that also increases the running time quite considerably even on some of our faster machines.

Best wishes

Christian

Am 31.10.2016 um 16:46 schrieb Dorothee Liebschner:

...

Hi Christian,

OK, it looks you found a way to prevent the positive density by using "realistic" starting B values (of the order of that of surrounding protein atoms). Did you also try to increase the number of macro-cycles?

Best wishes,

Dorothee

On Mon, Oct 31, 2016 at 9:11 AM, Christian Roth mailto:[email protected]> wrote:

Hi Dorothee

to answer your questions

The resolution is about 2.0 Ang and the average B around 55. The Bfactor from the ligand was set to the default of 20 by Coot when placing the ligands.

It seems that setting the B-value to the average B of the structure nearly solves the problem (most of the ligand fine, just parts with unusuallly high values). Setting it to a bit higher values 70 for a start and the ligand refines fine.

I noticed that in the first macrocycle with and initial factor of 20, the B's are nearly unchanged (20 +- 2) Red diff density appears as expected. In the second cycle The minimizer let the B-factors "explode" to about 200 and that basically is the overkill and it never recovers.

HTH

Cheers

Christian

Am 31.10.2016 um 15:59 schrieb Dorothee Liebschner:

...

Hi Christian,

Could you answer the following questions:

- what is the resolution of the data set? - what is average B of the protein? - what are the starting B values for the ligand? - did you try lowering starting B for the ligand and repeat the refinement? Is this behavior reproducible with different refinement options?

Best wishes,

Dorothee

On Sun, Oct 30, 2016 at 11:06 AM, Christian Roth mailto:[email protected]> wrote:

Hi Phenix Team,

regarding that ligand issue in this thread, we observed the same with a ligand in one of our structures now. After refinement the ligand is surrounded by pos diff density and the B-Factors are around 200 (way above any useful range in that case).

The cif was generated using elbow and phenix runs with options Real space refinement, TLS on, individual B, and optimizing weights. Nothing crazy.

Could you find a solution for the previous case and give us some tips?

Cheers

Christian

Am 06.09.2016 um 01:44 schrieb Pavel Afonine:

...

Hi Tongqin,

I second Dorothee's point that we need more information to resolve this problem. If you still have this problem please send me inputs files (data, model, .eff from last refinement, as well as ligand .CIF files if any) and I will look into this once I have files.

Pavel

On 8/31/16 09:29, Dorothee Liebschner wrote:

...

Hi Tongqin,

It is a bit difficult to diagnose with the information you provided. Could you please answer the following questions?

- Did you also try to reset the B-factors to similar values than neighboring atoms? F.ex. the average B-factor in the model could be 50 A**2, but in the ligand region, it could be lower, let's say 20 A**2. Then the starting value using average B is quite far from the likely B-factor of the ligand.

- What refinement strategy do you apply? How many macro-cycles? Do you use any non-default parameters for B-factor refinement?

- Which Phenix version are you using?

Best wishes,

Dorothee

On Wed, Aug 31, 2016 at 7:12 AM, Zhou, Tongqing (NIH/VRC) [E] mailto:[email protected]> wrote:

Dear All,

I am refining a structure with diffraction to 2.2A resolution and 95 % overall completeness. Now the Rs are at 18% and 23%, respectively. But the program failed to refine B factors for several ligands, atoms had high B factors while showing positive Fo-Fc map around them. Occupancy was set to 1 and I also tried to reset B to mean B of the whole PDB. Any of you seeing this phenomenon? Thanks!

Shown below is HEPES that has high B factor and positive density around it after refinement:

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

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

---

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