JISCMail - CCP4BB Archives

I think that first item on your possible to-do list is to verify that you have indeed crystallized the protein you purified. We, too, got great crystals once with protein X (100 kD) and noticed that 1) the lattice constants, space group symmetry, and Matthew's coefficient were within expected values (100 kD monomer in the ASU with moderate solvent content) and 2) the protein seemed to be cleaved into 50 kD fragments in the drop and in the crystal (as expected). However, it was a totally different protein that crystallized, which was why MR didn't work at all. After solving the structure by MIR, we found that a 50 kD minor contaminant (< 1%) crystallized, while our target protein did not. While there is still a good chance that the crystals you looked at contains at least a fragment of your target protein, make sure first, if you can.

As Matt recommended, analyze your protein by mass spec and/or N-terminal sequencing to verify that it is what you think it is. Then, as he recommended, try cloning and expressing a truncated variant. Careful limited proteolysis of the full-length protein would also be worthwhile in getting crystals faster.

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R. Michael Garavito, Ph.D.

Professor of Biochemistry & Molecular Biology

603 Wilson Rd., Rm. 513

Michigan State University

East Lansing, MI 48824-1319

Office: (517) 355-9724 Lab: (517) 353-9125

FAX: (517) 353-9334 Email: rm [log in to unmask]

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On Jun 20, 2012, at 2:13 PM, sonali dhindwal wrote:

Dear All,

I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good.

I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure.
Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%.

I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal.

I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions.

I will highly appreciate all the suggestions for this kind of problem.

Thanks and regards

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Sonali