Hi James, Can you, or anybody else, point me to a publication where "...not that uncommon for one or more crystal symmetry operators to "collapse" upon cryo-cooling..." Thanks, Nukri
Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 [email protected] -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of James Holton Sent: Thursday, September 30, 2010 8:43 AM To: [email protected] Subject: Re: [ccp4bb] difficult P1 crystal Yes, this sort of thing happens a lot more often than one might think, but people who have crystals with such "high-copy asymmetric units" tend to not solve them. Hence, they don't end up in the PDB. In cases where the structure is eventually solved, it is usually done by finding an alternative crystal form. At least, that is what I usually see when people bring these things to the beamline. Yes, the high NCS sounds like it would be really cool, but the reality of high-copy ASUs is that they are particularly prone to radiation damage problems. Not because of any special chemistry, but because the big ASU means that a complete data set from a single crystal requires collecting a lot of photons (see Holton & Frankel, 2010, and http://bl831.als.lbl.gov/xtalsize.html), and that high-copy ASUs tend to also have non-isomorphism "issues" (making it difficult to merge data from many crystals). Yes, the NCS advantages "cancel" this hindrance, but only at a much later stage (after you have found all the sites). That, and I think there is some psychological barrier to building and refining 24 copies of the same thing! Why do high-copy ASUs happen? Sometimes they occur naturally, like certain invertebrate hemocyanins where one molecule contain dozens to hundreds of copies of a single domain. Bart Hazes can tell you all about these! However, it is also not that uncommon for one or more crystal symmetry operators to "collapse" upon cryo-cooling (or other forms of crystal abuse). I have seen this a lot! There are many examples of nearly-crystallographic NCS in the PDB, many of which I suspect are cryo-cooling artifacts. Doesn't change the structure all that much, but should you choose to "go with it" you do have to be VERY careful with NCS like this! It is very easy to invalidate the Rfree. An extreme example is taking a crystal that is actually P2, but instead processing it as P1, picking a random "free" set, and refining with a twofold NCS operator. You will find that Rfree will drop like a rock and become essentially equal to Rcryst. This will be the case even if the structure you are refining is totally wrong! This is because every "free" reflection actually has an "NCS symmetry mate" in the working set. There are an alarming number of cases like this in the PDB, but I will not name names here. What is the "result" that makes you think your crystal is not tetragonal? -James Holton MAD Scientist On 9/30/2010 4:54 AM, Mario Milani wrote: > Dear all, > i have a 30 kDa protein that crystallize so far in three different conditions but with the same space group. It initially looks like tetragonal (I4, a=141, b=141, c=208) and then results triclinic (P1, a=141, b=141 c=144, alpha=119, beta=119, gamma=90), hosting about 24 mol. in the unit cell. Other data: self rotation shows the presence of 4 peaks with chi=180; molecular replacement shows the presence of a pseudo-translation peak; DLS made at protein concentration close to crystal growth conditions shows a Rh compatible with something like a tetramer with low polydispersity (about 15%). Do you have any experience with similar 'asymmetric' associations? Do you have any suggestions, beside the addition of ligands to the crystal growth conditions, in order to get a 'simpler' crystallographic assembly? I have some models (with sequence identity less than 25%) in order to try MR but all trials so far did not solve the structure (using balbes, molrep, phaser and epmr). Any suggestion is welcome. > Thank you, > > Mario Milani
