Re: [ccp4bb] Correct MR solution

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: arp/warp
From: Rafael Counago rafael {- dot -} counago {- at -} OTAGO {- dot -} AC {- dot -} NZ
Date: 2010-03-16

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From: Li Chan chanlixyl {- at -} HOTMAIL {- dot -} COM
Date: 2010-03-16

Subject: Re: Correct MR solution
From: Herman {- dot -} Schreuder {- at -} SANOFI-AVENTIS {- dot -} COM Herman {- dot -} Schreuder {- at -} SANOFI-AVENTIS {- dot -} COM
Date: 2010-03-16

Dear Bob,

I am quite puzzled by the results you describe. Here are some of my questions/comments:

- You could index in P2 and in P222. I assume you have in both cases the same cell dimensions and for P2 beta very close to 90Â°?
- How do the diffraction images look like? I would check every 30Â° or so. Are there hints for multiple diffraction, smeared spots, anisotropy?
- Is the Rmerge in P222 the same as in P2, or is the Rmerge in P222 worse? This could give a hint of your space group.
- Did you run pointless? With poor data, it might not give the right answer, but it does give useful suggestions how likely the possible space groups are.
- I assume the Matthews coefficient you quote is for P222 with one molecule in the asymmetric unit? For P2 there would then be 2 molecules in the asymmetric unit. If your P2 solution consists of only one molecule, this could explain the poor R and R-free as well as the extra density you observe.
- In MR, you have to check all possible space groups: P2221, P2212, P22121 etc. However, phaser and I guess phenix will do this automatically for you if you if you give the right keywords.
- Did you get an R/R-free of 21/24% both with P222 and with P212121? This appears highly unlikely to me and would point to problems with your data. If you get it for only one of the two, this solution is most likely correct and your protein of interest would then be disordered in the crystals.
- Does the crystal packing of this solutions contain voids which are big enough to accommodate 30 kDa of your protein of interest?
- How does the electron density look like: is it continuous, or does it have many breaks in the main-chain?

To come back to your questions: you will know the correct MR solution only after you have solved the structure, so you might have to pursue different possible solutions in parallel. To proceed further, I would do all the tests mentioned above and really make sure you did not make a mistake somewhere. If the best solution remains P212121 with good Rfactors and good electron density, the sad conclusion might be that your protein of interest is not ordered and therefore not visible in your crystals. Modern maximum likelyhood programs like phaser and buster do a decent job in revealing missing electron density, so I would definitively carefully examine the voids in your maps, to see if hints of the missing structure show up.

Good luck!
Herman

________________________________

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bob lainer
Sent: Monday, March 15, 2010 3:12 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Correct MR solution

Dear ccp4bbers,

I need some suggestions regarding structure solution of fused protein complex.

After repeatedly failing to obtain crystals of a 45 kDa protein (mostly a-helical) we fused the protein of interest to another protein of known structure which is 30 kDa is size (mostly b-strands). Many fusions were made and we selected a fusion-protein (Ca. 75 kDa) that has a minimal linker between the two proteins for crystallization trials. Both partners in the fusion are active and function as expected of wild-type proteins.

Crystals of the fusion protein were obtained and couple of datasets was collected. Running crystals of the fusion protein after X-ray data collection on SDS-PAGE shows that we have a band that is running approx. 60 kDa instead of 75 kDa indicating some form of proteolytic processing during crystallization (there are no bands that are equal to the 30 kDa fusion partner of known structure). One of the crystals diffracted to 2.5Ã… resolution. We could index the data in P2 (as well as P222 and P212121) space group, the highest resolution shell was cut off at 2.5 Ã… using I/Ïƒ = 2.0 as a guide. The overall Rmerge of 15% and completeness of 99%. The Matthews coeff. is 2.32 with 43% of solvent content as calculated with the molecular wt. of the fused protein complex (75kDa). We used Phaser (AutoMR, Phenix) and Molrep and Amore (CCP4) for molecular replacement using the known structure as the search model (the 30 kDa protein). In P222 and P212121, the statistics after one round of refinement post-MR statistics are good (R-factor and R-Free of 21% and 24%, respectively) and the known protein molecule fits and packs well with no extra density for the protein of interest. In contrast, in P2, we can see the extra density between planes formed by the known structural model. The spacing between these planes is about 30Ã…. The statistics of one round of refinement post MR is very poor (R-factor and R-Free of 41% and 47%, respectively).

The questions we are interested in are:-

1) How do we determine the correct MR solution (P2 or P212121)?

2) How should we proceed further in case like this?

Thanks in advance for the suggestions.

Bob

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: arp/warp
From: Rafael Counago rafael {- dot -} counago {- at -} OTAGO {- dot -} AC {- dot -} NZ
Date: 2010-03-16

Next message:
Subject: Re: arp/warp
From: Li Chan chanlixyl {- at -} HOTMAIL {- dot -} COM
Date: 2010-03-16