Dear Kris Kristal (if this is your real name), As Juergen pointed out, you may suffer from a twinning problem. However I do not think that in your case the twinnig fraction will give any useful information, since the proteins of the dimer will be almost perfectly symmetrical and only the peptide might or might not be really twinned.
If your 3.0, 3.5 and 3.7 Angstrom data sets are from different crystals, your results might be explained by different twin fractions of the different crystals (asuming that peptide 1 is unique as you mention in the end of your email and not peptide 3 as you mention in the beginning. Since the higher symmetry space group C2221, gives the lower Rfree, I would tend to publish the structure in this spacegroup. At 2.2 Å I would also refine without the peptide and look if the resulting difference density shows hints of an asymmetric position of the carbonyl groups and of differences between the leucine and isoleucine side chains. Good luck! Herman -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kris Sent: Wednesday, January 26, 2011 1:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Using Se-labelled peptide to 'deconvolute' pseudo-symmetry Hi everyone, I have a tricky structure that has pseudo-symmetry. Before we publish the structure, we would really appreciate some valuable feedback from experts like yourself. I have recently solved a crystal structure (2.2 Angstrom) made up of a peptide bound to a dimer. If I solve the structure in C2221 (Rfree = 25%), the asu consists of: 1) Dimer A-A' + peptide 1 (on symmetry axis) 2) Dimer B-B' + peptide 2 (on symmetry axis) 3) Dimer C-D + peptide 3 However, if I solve the structure in P21 (Rfree = 28%), the asu consists of: 1) Dimer A-B + peptide 1 2) Dimer C-D + peptide 2 3) Dimer E-F + peptide 3 4) Dimer G-H + peptide 4 Our peptide sequence is almost symmetrical (e.g. LLLGGGRGGGIII) , thus it is possible for it to be on the symmetry axis. However, in order to determine if the peptide really binds in both directions, we decided to synthesise a Se-Met labelled peptide. (We mutated one of the residues that does not interfere with binding into Met.) Recently, we collected 3 SAD data sets (3.0, 3.5, 3.7 Angstrom, 360 degrees each) of the Se-Met labelled peptide bound to the dimer. We processed the data in both P21 and C2221, then generated the anomalous difference map, using the above 2.2 Angstrom solved structure. The results are as follows: P21, 3.0 Angstrom Peptide 1: 2 anomalous signals (sigma level set to 3.0) --> Forward and reverse direction Peptide 2: 2 anomalous signals Peptide 3: 1 anomalous signal --> Forward direction only Peptide 4: 1 anomalous signal --> Forward direction only P21, 3.5 Angstrom Peptide 1: 2 anomalous signals Peptide 2: No anomalous signals Peptide 3: 1 anomalous signal --> Forward direction only Peptide 4: 1 anomalous signal --> Forward direction only P21, 3.7 Angstrom Peptide 1: 2 anomalous signals Peptide 2: 1 anomalous signals --> Forward direction only Peptide 3: 1 anomalous signal --> Forward direction only Peptide 4: 2 anomalous signals C2221, 3.0 Angstrom Peptide 1: 1 anomalous signal --> Forward direction only Peptide 2 & 3: (on symmetry axis) C2221, 3.5 Angstrom Peptide 1: 1 anomalous signal --> Forward direction only Peptide 2 & 3: (on symmetry axis) C2221, 3.7 Angstrom Peptide 1: 2 anomalous signals Peptide 2 & 3: (on symmetry axis) As the results do not consistently show that the peptide binds in both directions, we are now in a dilemma. How should we publish this structure and in which space group, since the structures in both space group refine very well with good statistics? Thank you in advance for all your suggestions and advice. Yours sincerely, Kris
