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Dear Jenny, I think it is almost impossible that you have only one molecule in the asymmetric unit. Assuming space group R32 with a=137 and c=87 A and a molecular weight of 10 kDa, you would have a solvent content of 84%. My guess is that you have 3 or 4 molecules in the asymmetric unit corresponding to 53 and 37% solvent content, respectively. Did you calculate a self rotation function to see if you get any information about how the molecules may be oriented? Did you calculate a native Patterson map to see if you have translational symmetry? Manfred. ******************************************************************** * * * Dr. Manfred S. Weiss * * * * Team Leader * * * * EMBL Hamburg Outstation Fon: +49-40-89902-170 * * c/o DESY, Notkestr. 85 Fax: +49-40-89902-149 * * D-22603 Hamburg Email: [EMAIL PROTECTED] * * GERMANY Web: www.embl-hamburg.de/~msweiss/ * * * ******************************************************************** On Wed, 17 Jan 2007 [EMAIL PROTECTED] wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > With these statistics, the solution is surely correct. > I would guess the problem is that you have somehow made a mistake in > applying the solution rotation and translation parameters to the > coordinate file to generate the pdb-file of the solution; perhaps you > applied them to the wrong file? Amore generates a transformed file > before starting (moved to the origin). Try applying the solution to > both your original file and the origintransformed one and see if one > of these gives reasonable Rs when refining. > Mark > > Quoting Eleanor Dodson <[EMAIL PROTECTED]>: > > > First of all - that looks a very clear solution - the first 4 peaks are > > all symmetry quivalents.. > > I would expect your problem is in the refinement. > > What resolution is your native data? > > > > Eleanor > > PS - and of course you can try MOLREP and PHASER and check they give > > the same solution.. > > > > > > Jenny wrote: > > > >> *** 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 > >>> > >> > >> > > >
