Re: [ccp4bb] mosflm and APS BM17
Hi I'd guess from the input file that the beam centre may be defined in a different reference frame to that which Mosflm expects in the image header; other possibilities are that the distance, wavelength, or something else (!) is wrong. Indexing is critically dependent on having the beam centre, distance and wavelength all being pretty close to the true values, though some indexing routines allow more latitude than others. My understanding (IMWBW) is that other programs (such as XDS or HKL2000) don't use the information in the image headers, and expect the parameters to be supplied by the user. With Mosflm, we try to make the user's life easier by trusting this information, especially from images collected at synchrotron beamlines; sometimes this trust is not warranted, and the user has to intervene! processing data collected on APS beamline BM17 on a MAR165 detector causes unexpected troubles. Even though the crystals are well-diffracting and the spots are sharp and well-resolved, indexing only works with a certain selection of frames, the refinement the goes totally hairwire. Probalby just a parameter or so set wrong. My input file in the attachment. The last three lines do not make much of a difference. Any ideas? Thanks! Cheers Jan mosflm.in: detector marccd directory ../Images template Image_0###.img image 001 nullpix 1 separation 0.95 0.95 close !LIMITS XMIN 0 XMAX 165 YMIN 0 YMAX 165 xscan 2048 yscan 2048 !SIZE 2048 2048 !PIXEL 0.07934 Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
[ccp4bb] crystal with precipitation
Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar
Re: [ccp4bb] crystal with precipitation
Oh, there is do much you can do! Lots of alternatives: 1. Decrease or increase the protein concentration (with a corresponding increase or decrease of precipitant. 2. Use a temperature gradient. 3. Set up in a capillary with liquid-liquid diffusion 4. SEED! #3 or #4 are your best bets, probably. For the capillaries, using Hamilton syringes: Pipet 20 ul protein in a 50 ul capillary. Ease it away from the end. When you are done, make sure you can reach the liquid with the syringe from the other side. Hold your finger on one end. Seal the other end with sticky wax Layer 5 ul water on top. Carefully layer the precipitant (10-30 ul) on top of the water. You might want to use a 4mM glass capillary mounted on a syringe, instead of Hamiltons. Seal the other end. Wait 3 days. Then look. Naturally, you can scale this down using 20 ul capillaries, or even smaller X ray capillaries. But then you can't use syringes to load them, use smaller capillaries. On the upside, you could shoot directly from the experiment. This has worked well for me. Lisa -Original Message- From: CCP4 bulletin board on behalf of shivesh kumar Sent: Wed 8/22/2007 3:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal with precipitation Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar
Re: [ccp4bb] mosflm and APS BM17
Hi all, thanks a lot for all the responses! As anticipated, the solution is rather simple - the origin of the beam: denzo refines to: x=82.6 y=80.3 mosflm reads from header: x=80.0 y=84.0 - bad indexing etc mosflm works with: x=82.6 y=80.3 No swapping of x and y between denzo and mosflm in this case. Cheers Jan On 8/22/07, Harry Powell [EMAIL PROTECTED] wrote: Hi I'd guess from the input file that the beam centre may be defined in a different reference frame to that which Mosflm expects in the image header; other possibilities are that the distance, wavelength, or something else (!) is wrong. Indexing is critically dependent on having the beam centre, distance and wavelength all being pretty close to the true values, though some indexing routines allow more latitude than others. My understanding (IMWBW) is that other programs (such as XDS or HKL2000) don't use the information in the image headers, and expect the parameters to be supplied by the user. With Mosflm, we try to make the user's life easier by trusting this information, especially from images collected at synchrotron beamlines; sometimes this trust is not warranted, and the user has to intervene! processing data collected on APS beamline BM17 on a MAR165 detector causes unexpected troubles. Even though the crystals are well-diffracting and the spots are sharp and well-resolved, indexing only works with a certain selection of frames, the refinement the goes totally hairwire. Probalby just a parameter or so set wrong. My input file in the attachment. The last three lines do not make much of a difference. Any ideas? Thanks! Cheers Jan mosflm.in: detector marccd directory ../Images template Image_0###.img image 001 nullpix 1 separation 0.95 0.95 close !LIMITS XMIN 0 XMAX 165 YMIN 0 YMAX 165 xscan 2048 yscan 2048 !SIZE 2048 2048 !PIXEL 0.07934 Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
[ccp4bb] [Fwd: Re: [ccp4bb]: self rotation function matrix NCS matrix]
Hi Eleanor and others: This is a little too late to follow up on this thread. But here is a similar question concerning MR and self-rotation. Without going into the translational part of MR, is it possible to tell the orientation of a 2-fold axis directly from the cross-rotation peaks? e.g. I have 2 top peaks from the cross-rotation function. There is also a strong 2-fold axis shown in self-rotation (kappa=180). Persumbaly then the two cross-rotation peaks are related by this 2-fold axis. From the angles of the two cross-rotation peaks, can I already tell the direction of the 2-fold axis? Regards, Weikai ÍúÍú wrote: I calculated the self-rotation function matrix with POLARRFN (named 1), and also got the molecular replacement solution with phaser, and used CNS's get_ncs_matrices to acquire the NCS matrix (named 2). Now what I wanted to do is to compare the results of 1 and 2. Are they the same or not, or which self-rotation function matrix is the right one of true NCS. Could you give me some suggestions? By the way, if I have got one molecule's PDB, and the rotation function matrix, are there any softwares on acquiring the other molecule's PDB? Many thanks POLARRFN will give you ALL symmetry equivalent rotations for your point group. Only one of these will relate your molecule A to B, and its inverse should relate B to A. Both will be listed in the POLARRFN output, plus probably many others depending on your point group.. I do not know the CNS convention now but it certainly used to be different from the CCP4 one. Hwever if you use he GUI cordinarte utilities superpose molecules select superpose by numbered residues then match A to B the log file will give you the ALPHA BETA GAMMA and the polar angles nt e same convention as POLARRFN. Eleanor
