Re: [ccp4bb] ITC thermogram
Probably, you have two mode binding between your ligand and protein. First time, when you had approximately 7 times molar excess of ligand, you could not monitor the endothermic reaction. When you went down to approx 2.5x lower conc of ligand, you could monitor endothermic reaction. However, what is Kd of the reaction? Is it very strong binding? How much micro cal values you are getting? When you dont see saturation, you increase the conc of ligand, why did you go down?
Re: [ccp4bb] ITC thermogram
Hi, I think the ligand concentration (250nM) is too low for the titration to reach saturation. To get properly saturated thermogram people sugest to use the ligand concentration 15-20 times higher than the macromolecule. On reducing the the ligand concentration you said you have obtained endothermic thermogram. Have you checked if the dP values are comparable or not? Sometimes when using too low concentrations of both protein and ligand may result in giving this kind of results, since the signals for the binding reactions get over-rided by the noise of the experiment. Running a blank experiment may help you to conclude about this confusion. Madhuparna. -- Madhuparna Bose. Senior Research Fellow. Department of Biotechnology. IIT Kharagpur. West Bengal. India. On Mon, Dec 15, 2014 at 11:35 AM, sajid akthar b_sajid_...@yahoo.co.in wrote: Dear All I am running ITC experiment for my protein. At 250 nm Ligand concentration (protein was 40nm) I was getting exothermic thermogram. But it was not saturated. I reduced Ligand concentration to 15 nm and checked again. I got endothermic thermogram. I dont understand why I am getting both endothermic and exothermic thermogram for same protein and ligand. Can some one explain Thank you Sajid
[ccp4bb] Call for access to Synchrotron Beamline Facilities, 2015 - EMBL Hamburg, Germany
Call for access to Synchrotron Beamline Facilities, 2015 - EMBL Hamburg, Germany We announce a call for synchrotron beam time applications in biological small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX) for the period April - November 2015. The following EMBL/DESY beamlines at PETRA III are available: P12 (SAXS), P13 (MX), and P14 (MX). A detailed description of the three beamlines and links to the electronic beam proposal forms can be found at: http://www.embl-hamburg.de/services/access_infrastructures/index.html The deadline for submission of proposals is January 31st, 2015. The proposals will be evaluated by an external Project Evaluation Committee. Users will be informed of the results of their applications by March 16th, 2015. Access to the EMBL Hamburg facilities also includes assistance with crystallization, sample preparation and, in combination with an EMBL beamline visit, with sample characterization and optimization. Usage of the beamlines and biophysical facilities can in part be supported by the European Commission, Research Infrastructure Action under the FP7 project BioStruct-X (http://www.biostruct-x.eu/). For further general information, please contact our user office: Tel: +49 40-89902-111 Email: useroffice (at) embl-hamburg.de For specific information, please use the following email addresses: saxs (at) embl-hamburg.de (small-angle X-ray scattering) mx (at) embl-hamburg.de (macromolecular crystallography) spc (at) embl-hamburg.de (sample preparation and characterization)
[ccp4bb] I am struggling to compile fasta36
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] I am struggling to compile fasta36
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.owork_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 -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
As a user of many of these coupling devices, I would highly suggest buying your couplers pre-fabricated and avoiding many hours (and possibly days) of headaches trying to manufacture these with your own hands. Rigaku Reagents sells them: https://www.rigakureagents.com/p-516-wizard-cubic-lcp-kit.aspx Formulatrix sells them a bit cheaper: http://formulatrix.com/store/product.php?productid=27cat=4page=1 I have used both products and they both function well even after many uses and cleaning cycles in methanol. Cheers, Jim On Mon, Dec 15, 2014 at 11:09 AM, Thomas Cleveland thomas.clevel...@gmail.com 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 -- Jim Fairman, Ph D. Group Leader I - Crystallography Beryllium http://www.be4.com Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
Hi Thomas, I have been doing LCP for a while and I am using the teflon ferrules that come with the 250 uL RN syringes instead. So on one side you would have two teflon ferrules and the other side you would have the ferrule that is attached to the needle and another plastic ferrule. I have also had luck using a 100 uL teflon ferrule (you have to hollow it out a bit) for this purpose. We have a machine shop here that can do the ferrule removal. I didn't have much luck doing this on my own. Hope that helps, Jarrod Mousa Graduate Research Assistant University of Florida -Jarrod Mousa On Mon, Dec 15, 2014 at 2:09 PM, Thomas Cleveland thomas.clevel...@gmail.com 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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
correction: www.hamiltoncompany.com addition: The compression fittings at least used to be available from us.vwr.com part number 89187-0002. On 12/15/2014 11:48 AM, Daniel Anderson wrote: 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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
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
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
Art Robbins Instruments also sells a zero-length coupler and it works well. BR From: Aaron Thompson [mailto:aaron.a.thomp...@gmail.com] Sent: Tuesday, December 16, 2014 12:02 AM To: b...@hofkristallamt.org Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation 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= 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
