Hi, Miguel, After I did translation, I found one molecue that looks good.I mean, I tried in O to generate symmetry related molecules and there are no overlaps.Thenwhen I did the rigid body refinement, the Rfac is about 58% and couldn't get lower. How to calculate how many molecules in this space group?I was told that there is only one molecule and we didn't find the second solution.
Thanks. jenny On 1/16/07, Miguel Ortiz Lombardia <[EMAIL PROTECTED]> wrote:
-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Hi Jenny. You say that the refinement of the solution for the first body gave high Bs and Rfactors. I wouldn't worry much about the Bs now, but what kind of Rfactors do you get? They will be necessarily high because you would expect 3 molecules in your AU. By the way, did you check the self rotation function for if you can see a non-crystallographic 3-fold? Even if you don't use it, it will help you confirm you have 3 molecules in the AU (of course, you can have them without having the non-crystallographic 3-fold) Anyway, after that refinement, did you look at the map, can you see information in that map? I mean: differences with respect to the model, but still clearly the same fold, though some betas can be not quite in place, etc. Very important, can you see extra density for extra molecule(s)? You could also use molrep fixing the "refined" model and using it for the next round of searches. This has helped a lot in many cases: if the refinement happens to catch some differences with your original model. Also, having 2.3A, why don't you give that model to ARP/wARP? It may well complete a good part of the structure. I have solved MR in H32 starting with a very similar but not perfect model. I had no particular problem. There is, to my understanding, no difference (for the matter of difficulty) in using the H32 or the R32 setting. Good luck! Miguel Jenny escribió: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Hi,Dear all > > Thanks very much for all the replies to my previous message.I'll get a > wrap up later.This is actually a follow up because I was suggested to > provide more information, so here it is. > > I just got the peptide sequencing result and it clearly shows that the > crystal is my protein. Gel-filtration and 1D NMR showed that it is a > monomer and well-folded.The wild type protein is a beta sheet protein > and also a monomer(10kd), the space group is P43212, with cell unit > a=b=49, c=71,alpha=beta=gamma=90. My protein is a 7 residue mutation > (not continous, but all within one loop that connect two beta sheet ) > and the crystal grows in a different condition from the wt > condition,it is actually grown from precipitant. > > We used HKL2000 to index and integrate the data, looks it's pretty > clear that the space group is primitive rhombohedral space group.But > the program automatically choose the cell size to be > 137.200 137.200 86.682 90.000 90.000 120.000 ( hexagonal > cell ) > rather than a rhombohedral cell ( a=b=c,alpha=beta=gamma),(can I force > it to choose the rhombohedral cell, does this matter?) > > So start from here, we scaled the data and did MR with amore.We tried > 8-4 A and here is part of the translation log file > .... > Translation solutions sorted by correlation coefficients > TABLE Alpha Beta Gamma Tx Ty Tz Corr_F > Rfac Corr_I Pkcount Dmin > SOLUTIONTF1 1 103.50 23.50 156.50 -0.1858 0.3493 0.4384 21.5 > 51.9 24.8 1 18.6 > SOLUT_21 1 103.50 23.50 156.50 0.8142 0.3493 0.4384 21.5 > 51.9 24.8 1 18.6 > SOLUT_21 1 103.50 23.50 156.50 0.4804 0.6818 0.2719 21.5 > 51.8 24.9 2 18.6 > SOLUT_21 1 103.50 23.50 156.50 0.1459 0.0145 0.1052 21.3 > 51.8 24.7 3 18.6 > SOLUT_21 1 103.50 23.50 156.50 0.3011 0.6855 0.2719 18.0 > 53.5 19.2 4 10.6 > SOLUT_21 1 103.50 23.50 156.50 0.2922 0.6063 0.1855 15.8 > 53.7 17.4 5 5.6 > SOLUT_21 1 103.50 23.50 156.50 0.6891 0.3409 0.3842 15.7 > 54.6 17.3 6 4.7 > SOLUT_21 1 103.50 23.50 156.50 0.0237 0.0085 0.0507 15.6 > 54.7 17.1 7 4.9 > SOLUT_21 1 103.50 23.50 156.50 0.3560 0.6746 0.2176 15.5 > 54.7 17.1 8 4.7 > SOLUT_21 1 103.50 23.50 156.50 0.0252 0.0100 0.0198 14.9 > 54.4 16.1 9 4.2 > SOLUT_21 1 103.50 23.50 156.50 0.6924 0.3432 0.3533 14.9 > 54.4 16.0 10 4.2 > SOLUT_21 1 103.50 23.50 156.50 0.3583 0.6767 0.1867 14.9 > 54.4 16.0 11 4.2 > SOLUT_21 1 103.50 23.50 156.50 0.6926 0.3428 0.3184 14.1 > 55.2 15.9 12 3.4 > SOLUT_21 1 103.50 23.50 156.50 0.1460 0.0120 0.4808 14.0 > 55.2 17.3 13 4.4 > SOLUT_21 1 103.50 23.50 156.50 0.3517 0.6419 0.2190 13.7 > 54.4 15.9 14 8.9 > SOLUT_21 1 103.50 23.50 156.50 0.6845 0.3082 0.3858 13.6 > 54.5 15.8 15 8.9 > SOLUT_21 1 103.50 23.50 156.50 0.6932 0.3449 0.2820 13.6 > 55.9 15.0 16 5.5 > SOLUT_21 1 103.50 23.50 156.50 0.0180 0.9748 0.0524 13.6 > 54.5 15.8 17 8.9 > SOLUT_21 1 103.50 23.50 156.50 0.0272 0.0123 0.4487 13.5 > 55.8 14.9 18 5.6 > SOLUT_21 1 103.50 23.50 156.50 0.0200 0.9767 0.0211 12.4 > 54.9 14.6 19 6.0 > SOLUT_21 1 103.50 23.50 156.50 0.6852 0.3092 0.3545 12.4 > 55.0 14.7 20 5.7 > .... > > This solution looks oK, so we tried to find a second structure, but > didn't succeed.Then we decided to go ahead to do refinement with this > structure first, because it's also possible that there is only one > structure in each asu.But the problem is , the B factor and Rfac is > always very high, in another word, we couldn't succeed for the next > step of refinement.Oh, forget to mention, we also checked that it's > not a twin. > > We also tried different resolution range, like 15-6, 8-3.5 and 10-5 > and didn't find the solution.Here's our concern: does this mean the > mut structure conformation is very different?Is that possible that > mutations in the loop disrupt the structure?Like domain swapping or > something like that.We actually also tried to cut down part of the > protein, but still, no luck. > > Right now, I don't know what to do next step, and I know some people > suggest me to try SeMet.I had a quite hard time with growing this > protein because it's only grow in very acid condition and I really > don't want to give up so easily with the data already in hand. > > Any suggestions what I could try? > > Thanks a lot for your help.I really appreciate it. > > Jenny > > > On 1/4/07, Jenny <[EMAIL PROTECTED]> wrote: >> *** For details on how to be removed from this list visit the *** >> *** CCP4 home page http://www.ccp4.ac.uk *** >> >> >> Hi, All >> >> I got a set of data of my crystal recently(2.3 A) and tried to do MR >> because it's only several residues different from the wild type. The >> problem is that after we indexed with HK:2000, it shows the primitive >> rhombohedral space group, with the cell size >> 137.200 137.200 86.682 90.000 90.000 120.000. >> >> The protein is only 90 residues ( 10kDa), so looks to me the cell size >> is a little big.But anyway, we tried to use the wt structure and do >> MR, didn't find the solution for the space group R32.We also tried C >> centered monoclinic space group and primitive triclinic space group, >> didn't get any luck either.Since the crystal is grown from the >> preticipate, so I started to worry that maybe the crystal isn't my >> protein.After I checked with SDS gel of the crystal, it shows very >> similar MW as the target protein.Now I'm waiting for the sequencing >> result. >> >> I heard that it's quite tricky for R32 and I wondered what's that.If >> the sequence comes back to be correct, what should I do the next >> step?If MR doesn't succeed, does this mean, the new mutant protein is >> very different from the wild type? >> >> Any suggestions would be appreciated. >> >> Thanks. >> >> Jenny >> > - -- Miguel Ortiz Lombardía Centro de Investigaciones Oncológicas C/ Melchor Fernández Almagro, 3 28029 Madrid, Spain Tel. +34 912 246 900 Fax. +34 912 246 976 email: [EMAIL PROTECTED] www: http://www.ysbl.york.ac.uk/~mol/ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Le travail est ce que l'homme a trouvé de mieux pour ne rien faire de sa vie. 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