Re: [ccp4bb] Peptide Crystallization
You could also consider organic solvents (or a mix) for crystallisation trials too. If you scan through the literature you will find that small peptides have in the past been treated as small molecules in terms of crystallization. Once the peptide gets over say 20 amino acids (not the exact number) then the peptides are treated more as small proteins for crystallography. On a side I did just see that the structure of Lysozyme in 50% ethanol was deposited in the PDB (not that lysozyme is a small peptide). Good Luck! Gina -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Buz Barstow Sent: Wednesday, May 25, 2011 8:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Peptide Crystallization Dear all, I am considering trying to crystallize a small peptide (around 15 amino acids). The peptide is soluble in neutral water or buffer (pH 7.0) until at least 10 mM (13 mg/mL), and adopts a turn conformation when bound to Zn. What are your thoughts on attempting this? If you think that it is worthwhile, what crystallization conditions would you try? I am thinking of a sparse matrix screen using the Hampton Crystal Screen 1 and 2 kits, using hanging drop crystallization in Hampton Vdx trays. Thanks! and all the best, ---Buz Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] glycerol
Hi Ray I have seen glycerol at less than 5% in the protein buffer prevent crystal growth completely and when removed from the buffer has resulted in very nice crystal growth of the glycerol free protein. Best Gina From: CCP4 bulletin board on behalf of Ray Brown Sent: Thu 10/03/2011 17:04 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] glycerol Hi all, I was intrigued by the recent question of whether glycerol had any adverse effects on the final purity of protein isolated by chromatography. Glycerol certainly helps to solubilize some proteins. Does anyone know of any negative effects of glycerol in protein purification, on protein crystal quality or use in cryocrystallography and on X-ray diffraction results? Cheers. Ray Brown Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Myristate
Hi there Sort of off topic... Does anyone know of a good resource/literature which demonstrates the pKa and other physical chemistry of Myristate and other fatty acids? Thanks for any info in advance Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] If it is a new structure?
Hi Liu Looks like (on the images you show) that you have a nice conformational difference for some of the helices between the 2 structures... Happy Hols Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Justin Hall Sent: Monday, December 20, 2010 8:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] If it is a new structure? Hi Liu, If I understand your question correctly, youre asking how different do two structures need to be for one to be new'. If by new you mean a new fold, then the answer is NO. Your structure and the homolog have the same fold. However, if your structure is the first structure of a protein in a new class, then your structure is a new insight for that reason (e.g. it is the first structure of a Unobtainium-metalloprotease). If it is not the first structure of a protein from a new class, lets say a previous structure of Unobtainium-metalloprotease has been solved using H. sapiens' sequence, but your protein is the first D. melanogaster ortholog solved, then your structure is a new insight for that reason. So, in a nut-shell, I guess what I am saying is that your protein is not a new fold, but is almost certainly new by some qualification, and you will know best what that qualification is. I hope that helps, cheers and happy holidays~ ~Justin On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Twinned data
Hi there Thank you for the advice regarding my twin data (mostly sent off the board). Seems I didn't put enough info in the email sorry for that! The resolution is 2.6Ang. My first thought was that the crystal was a fragment of the full length protein. The crystals are undergoing mass spec (the proteins itself appears stable using DLS etc)... I did try molecular replacement in the different space groups prior to detwin with variants of my search model. Truncate for the centric moments of E show a value close to 1.5 (perfect twin is 1.5) H test for twinning close -lkh is close to 0.5 Pseudo twin fraction is given as 0.43 The unmerged data processed in p1 are selected as p21 by Pointless and P421/3 if given as unmerged in p21. Thankfully I have new crystals now... Gina -Original Message- From: George M. Sheldrick [mailto:gshe...@shelx.uni-ac.gwdg.de] Sent: Friday, October 01, 2010 6:35 PM To: Clayton, Gina Martyn Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Twinned data If you really have the systematic absences for P41 or P43 then you must have (at least) four molecules in the cell, twinning cannot reduce this. Since a solvent content of 16% is too low, the most likely explanation is that it is not twinned but that the protein is smaller than the one you are expecting. With an unknown sequence and no experimental phase information such as SAD or MAD, your only hope would be Arcimboldo, but that requires data to about 2A or better (you don't mention the resolution). As an even longer shot, you could search the PDB for matching cells and space groups. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 1 Oct 2010, Clayton, Gina Martyn wrote: Hi there Just wondering if anyone has any suggestions how I can deal, if possible, with the following situation -. My first encounter with twinned data... which initially indexed as p43/p41 cell dimensions 58.4 58.4 61 90 90 90. Having seen the Matthews Coefficient for the solvent content of the unit cell as 16% I discovered the data are merohedrally twinned with twin fraction given as 0.1. I reprocessed the data as p1, p2 p21, c2221 (Rsymm etc indicates wrong space group, Mats Coeff 16%) p4 etc. In p2 the (pseudo) twin fraction is given as 0.43 (Mats Coeff 60% solvent 2.7 mol/ASU). I ran Detwin (ccp4) on the p2 p21 data with alternate operators as indicated by Xtriage in Phenix. I have had no molecular replacement solution i.e. Molrep rotational searches are not giving peaks and Phaser has not found a solution (nor with alternates of the search model). Does anyone have any suggestions/best paper to consult etc based on their experience of twinned data (aside from sort the crystals out...) or should I throw in the towell? Thanks in advance for any information and advice Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Twinned data
Hi Boaz thanks for the advice - Yes your suggestions are very useful thank you! I actually have not tried Phaser with the P4 data so I will give that a shot. Curiously Pointless selects P21 from P4 and P421/3 from P21... I tried MolRep in P1 but Phaser failed to find any solution though perhaps I did not pursue it enough... I think it is entirely possible that the space group I have , as in your example, may be something other than P21 etc Never tried Zanadu sounds interesting I'll take a look... We have new crystals... I was wanting to solve the twinned data to get some initial info etc and as a crystallography problem/exercise not having come across it yet... thanks again Best Gina From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il] Sent: Friday, October 01, 2010 5:18 PM To: Clayton, Gina Martyn Subject: Re: [ccp4bb] Twinned data Hi Gina, I've had a similar situation and here are some suggestions/ ideas: 1) You could try to run Phaser on the the data processed as P4 BEFORE detwinning and ask it to try all the possible space groups including the P422 and related ones. 2) Before step 1 or in parallel or following it, I would run the P4 processed data (again NOT detwinned) through pointless and/or xtriage to see whether they can give some indications for better choice of space group. 3) In my case, only running Phaser in P1 gave me a solution (the data were initially processed as P4 but I reprocessed them in P1 for that purpose). It's worth you trying too. 4) Eventually, I ran the P1 model (8 mols.) and data in Zanuda (on the York site) and that gave me a new space group choice (P21212). When I ran Phaser in this space group (after reprocessing the data again) it also gave a good solution which I'm now refining (with twinning) either in Phenix or refmac but without detwinning, since the program do this for you internally (I'm sure about refmac and assume it's the same in Phenix). I hope you'll find some of this helpful. Cheers, Boaz - Original Message - From: Clayton, Gina Martyn gina_clay...@merck.com Date: Friday, October 1, 2010 21:28 Subject: [ccp4bb] Twinned data To: CCP4BB@JISCMAIL.AC.UK Hi there Just wondering if anyone has any suggestions how I can deal, if possible, with the following situation -. My first encounter with twinned data... which initially indexed as p43/p41 cell dimensions 58.4 58.4 61 90 90 90. Having seen the Matthews Coefficient for the solvent content of the unit cell as 16% I discovered the data are merohedrally twinned with twin fraction given as 0.1. I reprocessed the data as p1, p2 p21, c2221 (Rsymm etc indicates wrong space group, Mats Coeff 16%) p4 etc. In p2 the (pseudo) twin fraction is given as 0.43 (Mats Coeff 60% solvent 2.7 mol/ASU). I ran Detwin (ccp4) on the p2 p21 data with alternate operators as indicated by Xtriage in Phenix. I have had no molecular replacement solution i.e. Molrep rotational searches are not giving peaks and Phaser has not found a solution (nor with alternates of the search model). Does anyone have any suggestions/best paper to consult etc based on their experience of twinned data (aside from sort the crystals out...) or should I throw in the towell? Thanks in advance for any information and advice Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact informationfor affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Twinned data
Hi there Just wondering if anyone has any suggestions how I can deal, if possible, with the following situation -. My first encounter with twinned data... which initially indexed as p43/p41 cell dimensions 58.4 58.4 61 90 90 90. Having seen the Matthews Coefficient for the solvent content of the unit cell as 16% I discovered the data are merohedrally twinned with twin fraction given as 0.1. I reprocessed the data as p1, p2 p21, c2221 (Rsymm etc indicates wrong space group, Mats Coeff 16%) p4 etc. In p2 the (pseudo) twin fraction is given as 0.43 (Mats Coeff 60% solvent 2.7 mol/ASU). I ran Detwin (ccp4) on the p2 p21 data with alternate operators as indicated by Xtriage in Phenix. I have had no molecular replacement solution i.e. Molrep rotational searches are not giving peaks and Phaser has not found a solution (nor with alternates of the search model). Does anyone have any suggestions/best paper to consult etc based on their experience of twinned data (aside from sort the crystals out...) or should I throw in the towell? Thanks in advance for any information and advice Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] model refinement
Not sure if anyone has mentioned this but for example what sort of maps are you using for your refinement. Have you tried density modification, or simulated annealing and or omit maps. These can really be useful for finding parts of your model that are not consistent with the data. Good luck! Gina From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of atul kumar Sent: Friday, June 18, 2010 5:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] model refinement (corrections, apologies!) Dear all Thanks for your replies. And thanks to all those who noticed that I had posted wrong unit cell dimensions for P3221 (which I picked from the header of the template pdb). My space group is P3221 (unit cell 54.8 54.8 100.1 90 90 120). Data quality appears fine. I/sigI=4.9; Rmerge=0.088; Completeness=99.9 (outershell values are: 2.3; 0.333; 99.9 respectively) As mentioned above, I solved the structure by molecular replacement and there appears to be no twinning. There are no major unmodelled densities. A couple of small blobs are present, which I think may be glycerol or some other molecule from my crystallization buffer but I still need to model these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Thanks again. Any other suggestions/comments are still welcome. Atul On Fri, Jun 18, 2010 at 7:39 AM, atul kumar atulsingh21...@gmail.com wrote: Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Catalytic residues in active sites
Hi there I wonder if anyone can recommend a good review/paper describing crystal structures that show high energy residues in active sites. By that I mean residues that may be in a strained conformation and rotate between conformations, such that they may even switch into unfavoured ramachandran regions during their activity, but that the strained high energy state is relevant to the enzyme activity? If I get enough responses I will list them and post them back up. Thanks so much in advance Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] B factor Coloring in Pymol
Dear Everyone Slightly off topic... I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Thanks in advance for any help Best Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] B factor Coloring in Pymol
Hi Ron Mmmm... not really but thanks for the info:) I have my Bfactor column assigned in percent conservation so the B value ranges from say 30% to 100%. I want to colour my molecule accordingly. Pymol allows B factor colouring but seems only to allow 3 colour choices. It used to allow 4 colour choices say WYOR I was wondering if any one had an idea how I could trick Pymol into accepting say 4 colours. Sorry if I was not clearer in the previous. Thanks again advance. Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Rob Nicholls Sent: Wednesday, March 31, 2010 3:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] B factor Coloring in Pymol Hi, I'm aware of two ways to do this: by writing the colour as a word, e.g: color white, (mypdbfile//A/*/*) which will colour a whole chain. Or by using rgb colours, e.g: set_color newcolor0 = [1,1,1]; color newcolor0, (mypdbfile//A/2/*) which will colour individual residues (residue 2 in this case) Pymol should accept a script containing such lines. Hope that helps, Rob On 31 Mar 2010, at 15:00, Clayton, Gina Martyn wrote: Dear Everyone Slightly off topic... I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Thanks in advance for any help Best Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] B factor Coloring in Pymol
Hi Robert That seems like a great suggestion! I will give it a shot and let CCP4BB know if it works Thanks Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robert Campbell Sent: Wednesday, March 31, 2010 4:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] B factor Coloring in Pymol Hi Gina, On Wed, 31 Mar 2010 15:00:34 -0400 Clayton, Gina Martyn gina_clay...@merck.com wrote: I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Say, your b-factor values range from 0-10, and your object is called protein, then you could do: color white, protein color yellow, protein b 2.5 color orange, protein b 5.0 color red, protein b 7.5 If you want to automate that process, then you need to write a script that determines, the b-factor limits for the 4 colours based on the b-factors present in your structure. Cheers, Rob -- Robert L. Campbell, Ph.D. Senior Research Associate/Adjunct Assistant Professor Botterell Hall Rm 644 Department of Biochemistry, Queen's University, Kingston, ON K7L 3N6 Canada Tel: 613-533-6821Fax: 613-533-2497 robert.campb...@queensu.ca http://pldserver1.biochem.queensu.ca/~rlc Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Crystal rescue
MiTeGen have instructions on their website for using their system. In practice I usually put a little silicon grease at the base of the capillary as sometimes the capillaries fall off plus you need reasonable steady hand eye coordination to put the capillary over the loop...I have found MiTeGen RT system very useful. Good luck! -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: Wednesday, January 27, 2010 7:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue MiTeGen (www.mitegen.com) sells a RT device - plastic capillary to put over loop... I do not know how well it would work for a long data collection - but people here have used it to evaluate their crystals Ezra On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote: Zhiyi, You can use a thin cap over your cryo loop, just put a drop of mother liquor in the top, place over the loop and make it airtight at the base. Not sure who sells these things though, I guess you can make it from a capillary too. Then remove the cryo stream or put it at a temp above freezing, say 253K. Flip Zhiyi Wei wrote: Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally
[ccp4bb] Re Anions in Protein Structures
Hi CCP4ers As requested I have compiled the list of responses I got regarding anions in protein structures. I did not receive a huge number (wrong time of day perhaps..) but I do think that the list below covers the various aspects (specific examples as well as various studies) and was very useful and interesting (thanks!). In no particular order 1. Anion binding sites in protein structures.Chakrabarti P. J Mol Biol. 1993 Nov 20;234(2):463-82. 2. On the routine use of soft X-rays in macromolecular crystallography. Part IV. Efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths. Mueller-Dieckmann C, Panjikar S, Schmidt A, Mueller S, Kuper J, Geerlof A, Wilmanns M, Singh RK, Tucker PA, Weiss MS. Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):366-80. Epub 2007 Feb 21. 3. Structural effects of monovalent anions on polymorphic lysozyme crystals. Vaney MC, Broutin I, Retailleau P, Douangamath A, Lafont S, Hamiaux C, Prangé T, Ducruix A, Riès-Kautt M. Acta Crystallogr D Biol Crystallogr. 2001 Jul;57(Pt 7):929-40. Epub 2001 Jun 21. 4. Data mining of metal ion environments present in protein structures. Zheng H, Chruszcz M, Lasota P, Lebioda L, Minor W. J Inorg Biochem. 2008 Sep;102(9):1765-76. Epub 2008 May 28. 5. Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1. White MA, Mast N, Bjorkhem I, Johnson EF, Stout CD, Pikuleva IA. Acta Crystallogr D Biol Crystallogr. 2008 May;64(Pt 5):487-95. Epub 2008 Apr 19. 6. Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid). Pallarès I, Fernández D, Comellas-Bigler M, Fernández-Recio J, Ventura S, Avilés FX, Bode W, Vendrell J. Acta Crystallogr D Biol Crystallogr. 2008 Jul;D64(Pt 7):784-91. Epub 2008 Jun 18. 7. The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies. Irimia A, Ebel C, Madern D, Richard SB, Cosenza LW, Zaccaï G, Vellieux FM. J Mol Biol. 2003 Feb 21;326(3):859-73. 8. Is the bond-valence method able to identify metal atoms in protein structures? Müller P, Köpke S, Sheldrick GM. Acta Crystallogr D Biol Crystallogr. 2003 Jan;59(Pt 1):32-7. Epub 2002 Dec 19. Best Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Anions in protein structures
Dear CCP4ers Can anyone recommend their favourite paper, or review, that discusses anions in protein structures. Thanks in advance and Happy New Year Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Van der Waals contacts
Hi CCP4ers Perhaps I am hashing over old news.but We are having a discussion about Van Der Waals contacts and effective contacts i.e. the real distance of a VDW bump between say a CH and a CH group which sometimes is described as between a C and a C as i.e. 2x 1.6A and ending about 4A but not including hydrogen. Some programs list contacts, to say a ligand, as far as 6A apart and some of the simulation programs use that distance too for contacts for protein protein interactions. Does anyone know of a good paper that discusses the effective distance or has a comment on where a VDW force may begin and end or it's effective distance - though some say it never truly ends just approaches zero... G Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Lipid content/removal
Also MALDI-TOF can be used to help identify the lipid (with standards). I vaguely remember one would extract the lipid, from the protein, using organic solvents then apply that to the MS. When I did it (about 10 years ago) I used non plastic vials and a Hamilton syringe so as not to contaminate the sample with plastics. Hope that's useful! Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Soisson, Stephen Sent: Tuesday, July 07, 2009 2:01 PM To: Subject: Re: [ccp4bb] Lipid content/removal Activated charcoal (see serum albumin structure references) can remove fatty acids. Good luck- Steve -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Tuesday, July 07, 2009 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lipid content/removal Dear Crystallographers, does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Thanks for your consideration, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
