[ccp4bb] Help request: Failed MR using the same molecule as model

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Date: 2009-11-24

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Subject: Help request: Failed MR using the same molecule as model
From: Chao Quan cuq102 {- at -} PSU {- dot -} EDU
Date: 2009-11-24

Dear CCP4 community:

I am a beginner to crystallography and therefore my apologies if this question is too
simple.

Basically we obtained several crystal forms of the same molecule, which is a hetero-
trimer containing protein A(18kD), protein B(16kD) and a RNA segment(40nt or about
15kD).

We have solved the structure of one crystal form(form_1); its information is as follows:
space group = P 42 22;
unit cell = 126.514 126.514 76.766 90.00 90.00 90.00
resolution = 2.7A;
Rwork = 0.25, Rfree = 0.265;
solvent content = 60%;
Number of molecules per Asymmetric Unit = 1;
Data redundancy = 5;
Data Completeness = 94%;

I am now trying to solve the structure of form_2 crystal using molecular replacement. So
far the information I know about form_2 is as follows:
space group = I 422 or I 41 22;
unit cell = 180.096 180.096 152.530 90.00 90.00 90.00
(unit cell is about 4 times the size of form_1)
resolution = 3.3A; (which is low)
Number of molecules per Asymmetric Unit = 2 or 3;
Data redundancy = 4;
Data Completeness = 92%;
There is no twinning(as shown by Sfcheck);
As shown in"Analyse Data for MR", the first peak is 100 and second is 68.92; I am not
sure if this indicates translation in a Asymmetric Unit;

The problem is, I can not get a good solution by MR using Phaser (both I422 and I4122
are tried). When I searched for 3 molecules per Asymmetric Unit, Phaser did not give
solutions at all.

When I used 2/ASU instead, I was able to get some solutions, with typical statistics as
follows:
RFZ=14, TFZ=35.9, PAK=0, LLG=1036;
However, these solutions had high R values(like Rwork=0.59 and Rfree=0.58), which
indicated that they are not solutions at all. Still, I tried refinement using refmac5, but R
values did not go down even after 50 rounds; sometimes they even increased after
refinement.

Besides, the RMS values bond length, bond angle and chiral center were all 0 as show by
refmac5.

I tried limiting resolution range to 15-4A in Phaser, which did not help either.

Now I am completely stuck. Could anyone give me some advice? I know this situation is
very strange, because I am using the SAME molecule for MR but can not can a solution.

Thanks a lot,

P.S.
1) Both form_1 and form_2 crystals were grown using Selenomethionine-containing
samples. There are 3 Sel_Met in protein A and 1 in protein B.
2) A 10-aa internal segment of protein B is missing in the solved structure, which may
indicate high flexibility.

Chao

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Methylation of macromolecular complexes
From: Sean Seaver sean {- at -} P212121 {- dot -} COM
Date: 2009-11-24

Next message:
Subject: Re: Methylation of macromolecular complexes
From: mjvdwoerd {- at -} NETSCAPE {- dot -} NET mjvdwoerd {- at -} NETSCAPE {- dot -} NET
Date: 2009-11-24