*** 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
