On Sun, Jun 10, 2012 at 7:57 PM, wrote:

I have a dataset in P21221. The cell content analysis shows that there should be 5 monomers in asu. The gel filtration profile of this protein shows that it should form a tetramer.

I agree with Francis - try all possible space groups in the P222 point group.

I have a simulated structure as searching model. I try to use the whole model or backbone model, and different resolution cutoff. When the whole model is used, the llg of solution is negative. When the backbone model is used, the llg is about 160 when searching for 4 monomers and tfz is 7.4. But the following autobuild process could not lower rfactor than 50%.

It would help if we knew more about the properties of the model and data. What is the sequence identity of the closest homologous structure? Comparative models are often problematic, and those statistics aren't great. Also, if the resolution is poor, AutoBuild isn't going to be able to do much. This may be a case where MR-Rosetta is the solution, but I'd verify that you're running the MR search correctly (and that it has any chance of working to begin with) before trying that.

I notice online that it might be useful to run self rotation function first to find ncs operators before doing mr. Is this true and how can i do this?

I'm pretty sure CCP4 has a program to do this, but I don't think we have anything in Phenix yet. (I suspect it's straightforward to code, but I couldn't figure out the math when I looked at it briefly last year.) -Nat

Hi Fengyun, it sounds like a perfect candidate for Arcimboldo. Last week at Erice school I've seen Isabel solving quite a few structures that people could not solve before using conventional methods. The best way is to contact Isabel directly (as it is not too straightforward to use Arcimboldo in its current state) and it's likely she solves it. Pavel On 6/10/12 9:39 PM, [email protected] wrote:

Hi Nathaniel,

The crystal reflects to about 2 A. Phenix.xtriage indicates there's no twinning. The protein is only 72 aa long by de novo design and we have a simulated model. This protein should form a three-helix bundle like structure. But i am not pretty sure how accuracy the model is.

Right now i am using phenix.automr to do all the stuff. I don't have rosetta installed yet.

Thank you! Fengyun

Quoting Nathaniel Echols :

...

On Sun, Jun 10, 2012 at 7:57 PM, wrote:

...

I have a dataset in P21221. The cell content analysis shows that there should be 5 monomers in asu. The gel filtration profile of this protein shows that it should form a tetramer.

I agree with Francis - try all possible space groups in the P222 point group.

...

I have a simulated structure as searching model. I try to use the whole model or backbone model, and different resolution cutoff. When the whole model is used, the llg of solution is negative. When the backbone model is used, the llg is about 160 when searching for 4 monomers and tfz is 7.4. But the following autobuild process could not lower rfactor than 50%.

It would help if we knew more about the properties of the model and data. What is the sequence identity of the closest homologous structure? Comparative models are often problematic, and those statistics aren't great. Also, if the resolution is poor, AutoBuild isn't going to be able to do much. This may be a case where MR-Rosetta is the solution, but I'd verify that you're running the MR search correctly (and that it has any chance of working to begin with) before trying that.

...

I notice online that it might be useful to run self rotation function first to find ncs operators before doing mr. Is this true and how can i do this?

I'm pretty sure CCP4 has a program to do this, but I don't think we have anything in Phenix yet. (I suspect it's straightforward to code, but I couldn't figure out the math when I looked at it briefly last year.)

-Nat

Thank you for your advice! Fengyun Quoting "Machius, Mischa Christian" :

Besides what others said about the technical aspects of your molecular replacement endeavors:

Gel filtration is not the most accurate method to determine oligomeric status. Also, the content of the asymmetric unit may or may not have anything to do with the oligomeric status of a protein. So, best not to pay too much attention to that. First, try to find four copies, then another one, if there is still space and there is electron density indicating another molecule.

More importantly, designed structures often deviate significantly enough from 'real structures that molecular replacement becomes difficult or impossible. There is great satisfaction to be derived from a designed molecule being suitable as a search model in molecular replacement, but in the end you'd really want an independently determined structure. I assume that part of the motivation behind determining the crystal structure of your protein is to verify your design. If so, a completely independent structure would be desirable and would also convince any reviewer. Thus, I would recommend generating the seleno-methionine variant of your design (provided it contains any methionines) and solving it experimentally.

Best of luck! MM

On Jun 10, 2012, at 10:57 PM, wrote:

...

Hi everyone,

I have a dataset in P21221. The cell content analysis shows that there should be 5 monomers in asu. The gel filtration profile of this protein shows that it should form a tetramer.

I have a simulated structure as searching model. I try to use the whole model or backbone model, and different resolution cutoff. When the whole model is used, the llg of solution is negative. When the backbone model is used, the llg is about 160 when searching for 4 monomers and tfz is 7.4. But the following autobuild process could not lower rfactor than 50%.

I notice online that it might be useful to run self rotation function first to find ncs operators before doing mr. Is this true and how can i do this?

Thank you in advance! Fengyun

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

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Thank you very much! The ab initio method may be worth trying first. Fengyun Quoting Andrew Purkiss-Trew :

For this resolution and type of problem, generating a Selenomethionine derivative is probably overkill.

Firstly, you can try abinitio methods such as Archimboldo (http://chango.ibmb.csic.es/ARCIMBOLDO/), which can work on 2Angstrom data given a helical structure.

Secondly, try generating derivative datasets. So, for example, Potassium Iodide may bind to the helices and give good anomalous phase information even on a home source. SIRAS can then be used to solve the structure. You will need to spend some time getting a good derivative, but it is generally quicker and cheaper than producing SeMet protein.

Good Luck,

Andrew Purkiss. X-ray Lab Manager London Research Institute.

________________________________________ From: [email protected] [[email protected]] On Behalf Of Machius, Mischa Christian [[email protected]] Sent: 11 June 2012 14:19 To: PHENIX user mailing list Subject: Re: [phenixbb] question on molecular replacement

Besides what others said about the technical aspects of your molecular replacement endeavors:

Gel filtration is not the most accurate method to determine oligomeric status. Also, the content of the asymmetric unit may or may not have anything to do with the oligomeric status of a protein. So, best not to pay too much attention to that. First, try to find four copies, then another one, if there is still space and there is electron density indicating another molecule.

More importantly, designed structures often deviate significantly enough from 'real structures that molecular replacement becomes difficult or impossible. There is great satisfaction to be derived from a designed molecule being suitable as a search model in molecular replacement, but in the end you'd really want an independently determined structure. I assume that part of the motivation behind determining the crystal structure of your protein is to verify your design. If so, a completely independent structure would be desirable and would also convince any reviewer. Thus, I would recommend generating the seleno-methionine variant of your design (provided it contains any methionines) and solving it experimentally.

Best of luck! MM

On Jun 10, 2012, at 10:57 PM, wrote:

...

Hi everyone,

I have a dataset in P21221. The cell content analysis shows that there should be 5 monomers in asu. The gel filtration profile of this protein shows that it should form a tetramer.

I have a simulated structure as searching model. I try to use the whole model or backbone model, and different resolution cutoff. When the whole model is used, the llg of solution is negative. When the backbone model is used, the llg is about 160 when searching for 4 monomers and tfz is 7.4. But the following autobuild process could not lower rfactor than 50%.

I notice online that it might be useful to run self rotation function first to find ncs operators before doing mr. Is this true and how can i do this?

Thank you in advance! Fengyun

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