[ccp4bb] Postdoc positions in Yokohama
Post-doctoral positions are available in the Protein Design group at Yokohama City University. Projects include beta helical bacterial virulence factors and protein-based nanocomponents for semiconductor manufacture (in collaboration with Panasonic). See J. Biol. Chem. 280, 17339-17345 (2005) and Structure 14, 925-933 (2006) for more details. The group is well equipped with three R-Axis detectors, AUC, ample wet lab facilities including crystallisation robotics and ready access to ITC, CD, SAXS and NMR, and is conveniently located between SPring8 and the Photon Factory. Please visit our web site for more information: www.tsurumi.yokohama-cu.ac.jp/pdl/index.html Yokohama is a lively city with great food and excellent travel connections to the rest of Japan and Asia. Interested candidates with a molecular biological or crystallographic background are welcome to send a CV to: Jeremy Tame Protein Design Laboratory, Yokohama City University Suehiro 1-7-29, Tsurumi-ku, Yokohama 230-0045, Japan Tel +81 (0)45 508 7228Fax +81 (0)45 508 7366
Re: [ccp4bb] Merohedral twinning
1) Why do you think there is twinning? If you label a structure whose true spacegroup is H32 as having SG H3, then some of the twinning analyses report that this is consistent with perfect twinning.. I believe the graphs of the moments from TRUNCATE give a true indicator. Ditto the latest SFCHECK in ccp4-6.0.2 does a good analysis and usually gets it right 2) If there is twinning smetimes a smaller crystal shows less effect. 3) If you can solve it then it can be refined using SHELXL. MR works for twiinned data usually.. Eleanor Mark Mayer wrote: Hi, I'd greatly appreciate advice on how to proceed with trying to solve and refine a structure with nearly perfect merohedral twinning - or is this impossible? The SG is H3; most likely nmol is asu is 4 from Matthews coefficient - but not sure about this. Here are the twinning stats from phenix xtriage -- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | -- | h,-h-k,-l | M | 0.038 | 0.452 | 0.468 | 0.478| -- The data is for a novel ligand complex for which we have previously solved structures with different ligands in other space groups - the protein has two domains, and these may have moved compared to other structures. So what should I do? Try and solve by MR using detwinned structure factors? Can Refmac refine the twin fraction, or should I refine against detwinned mtz file? Or ... should we look for additional xtal forms, or try additives to see if we can reduce twinning? Thanks !!!
Re: [ccp4bb] Multiple nucleation
In addition to trying lower concentrations of PEG and protein, you might consider whether aggregation in your protein stock or vibration in your crystallization environment are contributing to the problem, and how they might be minimized. It might be obvious, but we have found that it is important for protein stocks to be filtered or centrifuged to remove aggregates immediately prior to setting up drops, and if the protein solutions have been sitting around for a few hours or a few days, they must be centrifuged again. We also had a stupid incident recently where conditions that had previously generated high quality crystals were suddenly producing thousands of tiny crystals instead, and the problem was traced to a clinical centrifuge that had been moved to an adjoining bench and was vibrating the crystallization shelf. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-2857 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jobichen Chacko Sent: Thursday, March 29, 2007 7:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Multiple nucleation Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi
Re: [ccp4bb] Multiple nucleation
I've found that crystallization in sitting drops under oil dramatically reduces the no. of nucleation events and increases the overall crystal size. Now, if the increased crystal size helps improve diffraction is something to be tested. Among the many other suggestions... Raji -Included Message-- Hai All, Sorry for the non Ccp4 question. I got very small crystals for a protein and I am trying to optimize the condition to improve the size of the crystal and reduce the number of nucleation. The crystals are coming from five to six conditions , all have PEG 3350 in common. Apart from PEG, the condition has Lithium sulphate and Bis Tris in one condition and Ammonium suphate and HEPES in another condition, while the third condition is Sodium malonate, Bis Tris and PEG. I am getting thousands of small crystals with some precipitation. I tried macroseeding and additive screen, but the crystals are not growing. Any suggestions are more than welcome. Thank you. Jobi -End of Included Message--
Re: [ccp4bb] Right Handedness of Density
Dear All, We are working with a DNA binding protein with 4 Zn sites in in ASU. Using SnB and then CNS we were able to get a map using MAD phasing and could visualize the density for the double helix of the DNA but it was a left handed spiral instead of the usual right handed one. The space group which yielded the result was P3(2). We tried to flip the sites and change the space group to P3(1) but we are not able to generate a sensible map. Any advice regarding the best way to proceed will be very helpful. The resolution is around 3.2. Thanks Ruchi
Re: [ccp4bb] Right Handedness of Density
Hello Ruchi, I know that when you use non-crystallographic averaging there is a possibility of the starting with positive density and ending up with negative, the enantiomorph, or the negative enantiomorph density. In order to shift back to the correct phases you can apply a simple formula. This is the math that i recall: positive structure alpha negative structure alpha + 180 enantiomorphic structure-alpha negative enantiomorphic structure -alpha + 180 The recollection comes from a paper in methods in enzymology that i don't recall the author or year of at the moment. I know your case has nothing to do with averaging but the math should hold true. In this case, you can switch between the enantiomorphs by multiplying the phases by -1. I don't know if this holds a solution to your case. It may be worth a quick shot just to multiply the phase column by -1. I may be far off base here but i think i am correct. This of course is assuming that you have the correct space group and that you have the enantiomorphic phases. If i am totally off on this, someone please correct me. I'm really sticking my neck out late on a friday, eh! Good Luck- todd -Original Message- From: CCP4 bulletin board on behalf of Ruchi Anand Sent: Fri 3/30/2007 5:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Right Handedness of Density Dear All, We are working with a DNA binding protein with 4 Zn sites in in ASU. Using SnB and then CNS we were able to get a map using MAD phasing and could visualize the density for the double helix of the DNA but it was a left handed spiral instead of the usual right handed one. The space group which yielded the result was P3(2). We tried to flip the sites and change the space group to P3(1) but we are not able to generate a sensible map. Any advice regarding the best way to proceed will be very helpful. The resolution is around 3.2. Thanks Ruchi This email was scanned with Mcafee's Anti-Virus appliance, but this is no guarantee that no virus exists. You are asked to make sure you have virus protection and that it is up to date.
Re: [ccp4bb] Right Handedness of Density
Hi, If you have density for the protein and the DNA and the density for the protein is correct and you see left-handed DNA density, then I suppose you are seeing Z-DNA? Doesn't seem like a problem of incorrect enantiomorph if your protein density is fine. You can pull out a Z-DNA structure from the PDB and see if you can get a fit into the density that you see and if it makes sense you can then try to build an idealized Z-DNA based on the sequence you used for crystallization. Regards, Debanu. Green, Todd wrote: Hello Ruchi, I know that when you use non-crystallographic averaging there is a possibility of the starting with positive density and ending up with negative, the enantiomorph, or the negative enantiomorph density. In order to shift back to the correct phases you can apply a simple formula. This is the math that i recall: positive structure alpha negative structure alpha + 180 enantiomorphic structure-alpha negative enantiomorphic structure -alpha + 180 The recollection comes from a paper in methods in enzymology that i don't recall the author or year of at the moment. I know your case has nothing to do with averaging but the math should hold true. In this case, you can switch between the enantiomorphs by multiplying the phases by -1. I don't know if this holds a solution to your case. It may be worth a quick shot just to multiply the phase column by -1. I may be far off base here but i think i am correct. This of course is assuming that you have the correct space group and that you have the enantiomorphic phases. If i am totally off on this, someone please correct me. I'm really sticking my neck out late on a friday, eh! Good Luck- todd -Original Message- From: CCP4 bulletin board on behalf of Ruchi Anand Sent: Fri 3/30/2007 5:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Right Handedness of Density Dear All, We are working with a DNA binding protein with 4 Zn sites in in ASU. Using SnB and then CNS we were able to get a map using MAD phasing and could visualize the density for the double helix of the DNA but it was a left handed spiral instead of the usual right handed one. The space group which yielded the result was P3(2). We tried to flip the sites and change the space group to P3(1) but we are not able to generate a sensible map. Any advice regarding the best way to proceed will be very helpful. The resolution is around 3.2. Thanks Ruchi This email was scanned with Mcafee's Anti-Virus appliance, but this is no guarantee that no virus exists. You are asked to make sure you have virus protection and that it is up to date.
