Hi Peter,
    After you have finished checking the data again for twinning, etc.
as suggested by Eleanor,
you might try the following:
1) Refine using the simulated annealing refinement protocols in CNS and
then check R-values and map quality. If you try that, you can also test
a few different starting temperature values and slow cooling/constant
cooling during the annealing procedure. Following this, you can generate
composite omit maps and also try Primer and Switch phasing (look up
RESOLVE manual) to try to reduce model bias and try to improve map
quality which may allow you to trace more of the model.

2) What is the sequence identity of the target compared to the
homologous search model? Have you already changed all the sequence of
the MR model solution to reflect your actual target sequence? This may
help to improve your R-values.

3) Seems that you may have several different homologous models to choose
from. You may re-try MR using different homologs and see which gives you
the best starting R/Rfree and select that and change as many amino acids
as you can to reflect your actual target sequence and then try
refinement again.

4) Use some homology modeling program and try to remove elements of
secondary structure from your MR solution that may potentially not be in
your actual target or may be unstructured (insertions, deletions, etc.)
   
It is not extremely unusual to get a MR solution using a dimeric search
model instead of the individual monomers. You may also have the R
values given the 40% completeness and also the fact that the search
model is of a homolog.
Hope some of this may help your case although it may still take cycles
of iterative manual building.

Else, since you have 60% of the model missing, you may just have to go
for experimental phasing. Since you have a starting MR solution and thus
initial partial phases, even if you can get one or more low/medium
resolution derivatives on your needle crystals, you can use difference
Fourier methods to supplement the partial MR phases with experimental
phases for a complete structure determination. Of couse, if you are able
to improve your crystals, you should have more robust crystals for heavy
atoms soaks.

Thanks,
Debanu.

Eleanor Dodson wrote:

> I guess I would go back to the data.
>
> Could there be twinning?
> Is the a pseudo translation vector which means there are
> systematically weak zones of data? ( hklview might show something
> strange)
>
> Rfree seems rather close to R to me for this resolution - maybe it
> should go up a bit?
> How many cycles have you done already? PHaser refines without taking
> FreeR into account I think..
> And is there density for the missing bits?
>
> Eleanor
>
> Peter J Stogios wrote:
>
>> Hi,
>>
>> Long-time reader, first-time writer to ccp4bb.
>>
>> I'm having a problem with a 3.0 angstrom dataset (2.8 if I push it).
>> These crystals were thin but very long needles and I could see
>> diffraction only at the synchrotron. Diffraction spots were very
>> very small but well defined. High level of radiation damage.
>> P2(1)2(1)2(1), no large unit cell axes. R-int 7%(14 in highest
>> shell), I/sigma 14(8), completeness 98%. Nothing weird so far and
>> the stats actually look good. I was ecstatic these tiny needles
>> diffracted at all!
>>
>> I expect 2 chains by Matthew's coefficient and 50% solvent.
>> Biological unit is a dimer, homologs are dimers, this protein
>> purifies as a dimer. I'm solving it by molecular replacement, using
>> Phaser, which will give a refine-able solution only when searching
>> with dimeric models from homologous proteins. I cannot get solutions
>> that make sense or give Z-scores higher than 5 when searching for two
>> chains. But that doesn't matter, I get a solution when I search for
>> a dimer that is able to refine.
>>
>> So far so good. Here's where I get stuck: refinement with Refmac
>> goes well until R/Rfree values of 32/35, but I cannot break this
>> barrier. In fact, building into positive Fo-Fc peaks results in
>> R-free getting worse--actually any further refinement at all results
>> in R-free going up. The model is only 40% complete and has many
>> missing regions, not only in loops in turns. I just can't improve
>> the model anymore. I've tried rebuilding in resolve, Arp/warp, and
>> of course lots of manual building.
>>
>> I don't think my data is as bad as to restrict my refinement, so I'm
>> confused as to why I've hit this barrier. Hopefully this description
>> isn't too vague but I appreciate any help in advance and I can
>> elaborate if you're willing to help!!
>>
>>
>> ~
>> Peter J Stogios
>> Ph.D. candidate, Privé Lab
>> Dept. of Medical Biophysics, University of Toronto
>> Toronto Medical Discoveries Tower (TMDT) at MaRS
>> 101 College St., Rm. 4-308
>> Toronto, Ontario M5G 1L7
>>
>> e: [log in to unmask]
>> w: http://xtal.uhnres.utoronto.ca/prive
>> p: (416) 581-8550 ext. 7543
>>
>>
>>
>