[ccp4bb] P31 or P32
Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
Hi Thomas We use a very simple solution. We simply shorten a normal needle to 6.5 mm and push it into another syringe that has *two *of the white PTFE ferrules made by Hamilton. (We salvage the extra ferrule from another needle.) We use a 22 gauge needle (not 22S, where the S stands for *small *inner diameter). Now you can push the two syringes together and make your LCP. It helps if you tighten the knurled nose-piece to compress the PTFE ferrules before you join the syringes. You have to keep a little pressure on the two syringes to hold them together while you mix up the LCP, or use eg tape. (Which is why we've made a little mixer on a rail, but that's another story.) One nice feature is that you hardly waste any LCP and the needle for dispensing it is already attached to the syringe. Let me know if you need more info or you'd like me to send you a short needle. Best wishes, Patrick On 16 December 2014 at 02:14, Thomas Cleveland thomas.clevel...@gmail.com wrote: Hi everyone, Thanks for the advice. I had, in fact, already ordered some commercial couplers, but they haven't come in yet, and there was an experiment I wanted to do today. Here's what I found: 1. The steel ferrules have an orientation, with a tight side and a loose side. They can be removed by sliding in one direction, but not the other. Even then, it's pretty difficult. I had to use pliers, and pieces of needle broke off during the process. The tight side of the ferrule then needed to be reamed open slightly with a steel tool before I was able to slide the ferrule onto the other needle. 2. Soldering stainless steel is really a pain. In the end I got a coupler that seems to work well, but it was a pain, and it's a bit charred looking. Thanks again, Tom On Mon, Dec 15, 2014 at 6:01 PM, Aaron Thompson aaron.a.thomp...@gmail.com wrote: I agree with Jim – purchasing the couplers will get you up and running much quicker. TTP also sells nice couplers: https://www.ttplabtechstore.com/ttp_ecom/cc/ItemDetails.jsp?@where.ItemID@EQ=3072-01050sessionkey=# Aaron On Mon, Dec 15, 2014 at 12:38 PM, Bernhard Rupp hofkristall...@gmail.com wrote: Tried the RN kludge at least I did not get it to work. You cannot tighten the plastic swage lock type sleeves tight enough. On operation the pressure drives the PEEK tubing out of the compression fit. Maybe if you have a jig that holds the 2 syringes in a fixed position so they cannot move apart it can work. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Daniel Anderson Sent: Monday, December 15, 2014 8:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation Here's my addition to Jim Fairman's reply: You could use a pair of RN compression fittings (www.hamilton dot com part number 55751-01) and a segment of HPLC tubing. HPLC tubing within my field of view can have an inside diameter as small as 0.005 inch. hope that helps, Happy Merry, etc., Dan On 12/15/2014 11:09 AM, Thomas Cleveland wrote: Hi all, I'm trying to put together some homemade syringe couplers following the published instructions from the Caffrey group. I'm having a bit of trouble with this part: The stainless steel ferrule of the second needle is removed and placed on the free end of the coupling needle such that the double thumb nut is held symmetrically between the two steel ferrules Has anyone done this, and if so, how did you remove the stainless steel ferrule from the second needle in order to place it over the first? The stainless steel ferrule appears to be firmly attached and I'm having trouble removing it. Thanks, Thomas Cleveland -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] P31 or P32
If your Pt position is centric then either hand will give a reasonable looking solution but one will generate LH helices and the other right. Model building can break that ambiguity - SHELXE and Buccaneer both will impose correct peptide geometry. But at such low resolution they may struggle.. but it is worth a try Eleanor On 16 December 2014 at 09:39, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys
Re: [ccp4bb] P31 or P32
What do the twinning tests show? Those space groups can be suspicious, I believe. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER Sent: Tuesday, December 16, 2014 4:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] P31 or P32 Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys
Re: [ccp4bb] P31 or P32
Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] P31 or P32
Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] I am struggling to compile fasta36
Dear Pietro, Tim's solution should work for you. If not please let me know. From CCP4 6.5.0 (due very soon), the default sequence search tool in MrBUMP will be Phmmer which will be included in the suite on all supported operating systems. So there won't be the need to download fasta separately. Best wishes, Ronan On 15/12/14 16:47, Tim Gruene wrote: Dear Pietro, zlib is the C compression library. You must have some version of libz.so installed in order to link againt it. Regards, Tim On 12/15/2014 05:36 PM, Pietro Roversi wrote: Dear all, I apologise for the dullness of the question, but I am struggling to compile the fasta36 binary that I need to run MrBump: emmaWT:/software/fasta-36.3.7/src sudo make -f ../make/Makefile.linux64_sse2 all gcc -g -O -msse2 -o ../bin/fasta36 comp_mthr9.o work_thr2.o pthr_subs2.o compacc2_t.o showbest.o build_ares.o re_getlib.o mshowalign2_t.o htime.o apam.o doinit.o init_fa.o drop_nfa.o wm_align.o calcons_fa.o scale_se.o karlin.o lgetlib.o lgetaa_m.o c_dispn.o ncbl2_mlib.o lib_sel.o url_subs.o mrandom.o -lm -lz -lpthread /usr/bin/ld: cannot find -lz collect2: error: ld returned 1 exit status make: *** [fasta36] Error 1 Googling for this -lz ld flag does not seem to hit anything immediately relevant in the top pages. Please help! Pietro Sent from my Desktop Dr. Pietro Roversi Oxford University Biochemistry Department - Glycobiology Division South Parks Road Oxford OX1 3QU England - UK Tel. 0044 1865 275339
Re: [ccp4bb] P31 or P32
Hi Eleanor, The data is perfectly twinned, with the original auto processing assigning a point group of P312, but I reprocessed it in P3 and it seems to be behaving okay. Are there any additional precautions I should take at the phasing stage with twinned data? BW Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 16 December 2014 15:13 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] P31 or P32
I would say try to get better crystals and data, and also do a MAD experiment. But...perfect twins are really hard, I think. Maybe vary the crystallization conditions a bit, additive screen, seeding, etc. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER Sent: Tuesday, December 16, 2014 10:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Hi Eleanor, The data is perfectly twinned, with the original auto processing assigning a point group of P312, but I reprocessed it in P3 and it seems to be behaving okay. Are there any additional precautions I should take at the phasing stage with twinned data? BW Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 16 December 2014 15:13 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] P31 or P32
But are you sure it is twinned and not really P312? Ltest etc is a pretty reliable indicator but the results can be biased by non-crystallographic translation, etc And do the HA sites obey the twinning symmetry? On 16 December 2014 at 16:36, Keller, Jacob kell...@janelia.hhmi.org wrote: I would say try to get better crystals and data, and also do a MAD experiment. But...perfect twins are really hard, I think. Maybe vary the crystallization conditions a bit, additive screen, seeding, etc. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER Sent: Tuesday, December 16, 2014 10:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Hi Eleanor, The data is perfectly twinned, with the original auto processing assigning a point group of P312, but I reprocessed it in P3 and it seems to be behaving okay. Are there any additional precautions I should take at the phasing stage with twinned data? BW Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 16 December 2014 15:13 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.demailto: t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] P31 or P32
I agree here. The 50 % could be a good indicator. I am finishing a new structure which was about 42% twinned in the monoclinic setting (b = ~90) for an Se-Met dataset. When I went to refine it with the native dataset, the twin fraction increased to 50%, figured something wasn't right. fortunately, the native was much better behaved / untwinned in an orthorhomic crystal form with a near identical unit cell. -tg From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: Tuesday, December 16, 2014 2:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 But are you sure it is twinned and not really P312? Ltest etc is a pretty reliable indicator but the results can be biased by non-crystallographic translation, etc And do the HA sites obey the twinning symmetry? On 16 December 2014 at 16:36, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: I would say try to get better crystals and data, and also do a MAD experiment. But...perfect twins are really hard, I think. Maybe vary the crystallization conditions a bit, additive screen, seeding, etc. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER Sent: Tuesday, December 16, 2014 10:37 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Hi Eleanor, The data is perfectly twinned, with the original auto processing assigning a point group of P312, but I reprocessed it in P3 and it seems to be behaving okay. Are there any additional precautions I should take at the phasing stage with twinned data? BW Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson [eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk] Sent: 16 December 2014 15:13 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P31 or P32 Sorry - of course it can.. Sorry. Only if the twinning is perfect then you apparently get a higher symmetry.. Eleanor On 16 December 2014 at 14:26, Tim Gruene t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.de wrote: Dear Rhys, I would try to place idealised secondary structure elements with coot into the density - at this resolution they probably fit both hands, but you may see a difference when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I run SAD phasing using Autosol in Phenix. After density modification I get pretty nice looking maps with clear solvent channels and interconnected density for protein, however there is very little difference in map R-factor between the hands and the maps from both the P31 and P32 solution appear comparable in structure. Obviously DM is struggling to break the hand ambiguity, however can some one tell me if what I'm seeing represents a definite solution? And what is the best way to proceed from here? Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Scientist and Postdoctoral Positions in Structural Biology
Several scientist and postdoctoral positions are available in prof. Zhiwei Huang’s laboratory at Harbin Institute of Technology (HIT), China. Our research interests focus on studies of the mechanisms of pathogen (such as HIV and Ebola virus)-host interactions, and the mechanisms of how signals transduce across the membrane by GPCRs and transporters. We use multi-disciplinary approaches including X-ray crystallography, Cryo-EM, virology, cell biology, and mouse genetics in our studies. We are equipped with a state-of-the-art facility to conduct soluble/membrane protein structural and functional studies including home X-ray source, an automated crystallization screening device, and other setups such as ITC. Highly motivated candidates with Ph.D. and background in macromolecular X-ray crystallography or cryo-electron microscopy are preferred, but enthusiastic individuals with a Ph.D degree in virology, cell biology, biophysics, physics or related fields are also encouraged to apply. We offer competitive salary and benefits, which will be commensurate with experiences. For further information, please vist the university website ( http://life.hit.edu.cn/News/Show_e.asp?id=4405). Applications with a statement of interest, a full CV and three contacts for reference should be sent to prof. Zhiwei Huang at huangzhiwei2...@gmail.com
