Re: [ccp4bb] Membrane Protein Crystallization Plates
Yuri I know this isn't quite what you're asking, but it's helpful to use the COC (UV permeable) version of plates if you're crystallizing samples that contain detergent. It's just that the drops tend to spread very thinly if you use the normal polystyrene PS plates. The COC is more hydrophobic. (On the other hand we prefer PS plates for normal proteins with no detergent.) Patrick On 25 February 2012 20:35, Yuri Pompeu yuri.pom...@ufl.edu wrote: Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri -- 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] Any calculation function in CCP4 to analyze coordinate file?
We have been doing a bit of this using a simple program under Linux - pdbskim. A linux binary available on request. Very simple output though, focused on infromation on occ, Bs alternatives, etc to help make decisions during structure refinement. Jan On Sat, Feb 25, 2012 at 11:59 PM, WENHE ZHONG wenhezhong.xmu@gmail.comwrote: Dear members, Just have a silly question past my mind: is there any program in CCP4 can be used to analyze coordinate file (.pdb format) to have a very general/overall discription about the structure? --such as the total number of protein residues/water/ligand, the total atoms of protein/water/ligand, the average b-factor of protein/water/ligand, the number of residues which have alternative side chians, and so on. Thank you. King regards, Wenhe -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
Re: [ccp4bb] Postdoctoral position in structure-function studies of enzymes for biotechnologies
Apologies - the limiting date for PhD title is different, as stated here: The applicant has received PhD degree after March 28, 2008. Jan Dohnalek IMC Prague On Sun, Feb 26, 2012 at 9:52 PM, Jan Dohnalek dohnalek...@gmail.com wrote: DEADLINE APPROACHING! Our group has been involved in structural studies of several novel enzymes interesting from the point of view of biotechnologies, such as Laccase from *S. coeliocolor, *beta-galactosidase from Arthrobacter (660 kDa) or recently plant metal-dependent bifunctional nuclease or bacterial organophosphate anhydrolases for removal of toxic warfare agents. To continue studies in this area and apply mostly structure-driven analysis on further targets we are still accepting applications for a postdoc position. The Institute of Macromolecular Chemistry of the Academy of Sciences (IMC) in Prague, Czech Republic searches for highly motivated young researchers for a full time research postdoctoral position in the area of *Structural Analysis of Biomacromolecules.* The position is offered within the Department of Structural Analysis of Biomacromolecules (http://www.imc.cas.cz/en/umch/o_struanal.html) where the central technique is x-ray diffraction analysis of mostly protein structures. *Topic A: Structure and function of enzymes for biotechnologies* *Structure-function studies of enzymes with biotechnological applications. * The work will be focused on defining macromolecular targets, protein production and purification in collaboration with a partner, crystallization, X-ray diffraction, determination and interpretation of structure, design of mutations and complementary biophysical techniques, including measurements of reaction kinetics, inhibition and complex formation. The successful applicant will be also involved in ligand selection/design and studies of complexes of such compounds with the target macromolecules. The applicant has received PhD or equivalent in one of the following fields: biophysics, physics, biochemistry, chemistry, molecular biology or closely related. Required experience and skills: protein crystallography, biochemistry, reaction kinetics, inhibition. Practical experience with protein-ligand interaction studies (ITC, SPR, etc.) and with enzyme modification/optimization is welcome. * * The applicant has received PhD degree after May 28, 2008. *Contract type: * Full time, fixed term, 3 years, starting 1st July 2012, EU funded via the Czech Ministry of Education. You can contact me on this e-mail address or send your full application according to instructions available here: http://www.imc.cas.cz/en/umch/aktuality.htm?id=97 The deadline for full applications is February 29! Jan Dohnalek IMC Prague -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410 -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
Re: [ccp4bb] Membrane Protein Crystallization Plates
Hi i do not want to get into trouble by going against any products The plates what you are talking about as soon it came out we tested. I did not look back to see if there was a monoolein:cholestrol coated plate. The ones i used were for sure monoolein coated ones. That tells it all.The rest of the plates still unused. I set up plates with bacteriorhodopsin even that did not work in our hands (the bacteriorhodopsin set up otherwise would work very well.) By the way it did not work for me did not mean it is bad. And the plates were in plasticware made it highly birifringent to document the results. But the plates Patrick says here i am not sure about. If Patrick want to to tell me a little bit more about i would like to hear that. I would set up plates the normal way. I am really not convinced mesophase will form by just adding protein-detergent to a previously poured monoolein. anyway i am also interested to hear experts views of the method adapted in this product. There is a poster that they shown at their website shows bacteriorhodopsin and another membrane protein crystallized using these plates. These are my personal opinion. Pius On Sat, Feb 25, 2012 at 3:35 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] coot with probe and reduce
Dear B,Yes its working. Thanks for your help. ThanksYogi Date: Sun, 26 Feb 2012 22:49:08 +0100 From: bernh...@chem.gla.ac.uk Subject: Re: [ccp4bb] coot with probe and reduce To: CCP4BB@JISCMAIL.AC.UK Dear Raj, please follow exactly what is written in the FAQ http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.html#mozTocId860510. I just updated it slightly to hopefully make things even clearer (*). You seem to be mixing different possible setups. Either you move the files to WinCoot\bin OR you specify them in other files. I assume you did all of the above and got confused. Hope this helps, B (*) and there was a typo that the path separators have to be \\ rather than \ (if you wish to specify the absolute file names). Dear All, I am trying to use probe and reduce with coot new version. I tried as suggested in coot FAQ page- 1. renamed probe.exe and reduce.exe were put in C:\WinCoot\bin- didnt show validate/probe clashes as active n coot 2. made changes to group settings.py in wincoot/share/coot/python folder to probe_command = C:\WinCoot\bin\probe.exe reduce_command = C:\WinCoot\bin\reduce.exe probe_command = probe reduce_command = reduce Any help is appreciated. Thanks raj No virus found in this message. Checked by AVG - www.avg.com http://www.avg.com Version: 2012.0.1913 / Virus Database: 2114/4829 - Release Date: 02/24/12
[ccp4bb] Desalting columns
Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sangeetha, provided you express the protein in E. coli, you could also sonicate the cell debris in various buffers and compare supernatant/pellet on SDS-gel. It might already give you a first clue and is faster, cheaper and you don't risk to clog the new columns with aggregated protein. Tim On 02/27/2012 05:01 PM, Sangeetha Vedula wrote: Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPS6xEUxlJ7aRr7hoRAqOqAJ0cYn4488ctcIRUeZa4K3f2smojfQCeKBDI +nO8deQmkPNB9nWSWJtBoOI= =SU1z -END PGP SIGNATURE-
Re: [ccp4bb] Desalting columns
I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. ~ 1 ml spin columns with Sephadex G25 or Biogel P6. Typically, when loading 200 ul of the sample onto 1 ml column, desalting efficiency is ~ 95% and protein recovery is ~ 90-95% without volume change. Lowering sample volume will increasing dealting and reduce yield but I never had the yeild worse than 75% even with 50 ul volume. Bio-Rad sells prepacked Bio-Spin P-6 or you can buy empty columns and dry P6 (by fine grade in this case) and pack to 1.2 ml per column, which will take your 250 ul volume and still result in decent desalting. Pharmacia probably sells something similar and many companies sell 0.5 ml spin columns. I like P6 (polyacrylamide) better than Sephadex (dextran) because usually (but not always) non-specific binding is lower. - Dima Thanks a lot! Best regards, Sangeetha.
[ccp4bb] Coot Crashed
Dear All: I am having trouble with Coot. The program keeps crashing when I click on rotamer analysis. Other functions, sych as geometry analysis all worked fine. It runs normal before, and only happened when I added the ligands into the model. I am using WinCoot_0.7_pre-1-revision-3772, and window vista. Thank you for advices. Ros
Re: [ccp4bb] Desalting columns
Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
[ccp4bb] ideal MG-oxygen distances in refmac
Hi all, I'm building a ~1.9Å structure that has a few Mg++ ions bound. I thought that the expected distance for Mg-O was 2.1Å, but in refmac the default to Asp/Glu oxygens appears to be 1.91 Å. Strangely, for a Mg-bound pyruvate ligand, the default distance to one oxygen is 2.18 Å, but 1.91 Å for the other: = = = (from log file) I am reading library. Please wait. mon_lib.cif INFO: link is found (not be used) dist= 2.006 ideal_dist= 1.910 ch:AA res: 272 GLU at:OE1 .-ch:Ab res: 601 MG at:MG . INFO: link is found (not be used) dist= 1.943 ideal_dist= 1.910 ch:AA res: 296 ASP at:OD2 .-ch:Ab res: 601 MG at:MG . INFO: link is found (not be used) dist= 1.853 ideal_dist= 1.910 ch:Ab res: 601 MG at:MG .-ch:Ac res: 602 PYR at:O2 . INFO: link is found (not be used) dist= 2.061 ideal_dist= 2.180 ch:Ab res: 601 MG at:MG .-ch:Ac res: 602 PYR at:O3 . INFO: link is found (not be used) dist= 2.076 ideal_dist= 1.910 ch:BB res: 272 GLU at:OE1 .-ch:Bb res: 601 MG at:MG . INFO: link is found (not be used) dist= 2.045 ideal_dist= 1.910 = = = What's the easiest way to change this default distance - altering a file in the monomer libraries or explicitly stating the LINK distances? I'm using refmac version 5.6.0117 in ccp4 6.2.0 on MacOS 10.6.8. Thanks, Greg
Re: [ccp4bb] Membrane Protein Crystallization Plates
Yuri, Did you mean plates for setting up Lipidic Mesophases? If so, here is a listing of products I have used in the past. I highly endorse the plates from Molecular dimensions, particularly the plastic laminex plates (MD11-51-100 + MD11-54). They do have the dis-advantage of drying out after a 2-month or so period, but they make the job of harvesting significantly easier than dealing with the glass equivalents (so much so that I don't ever plan on using the glass bases and covers again). *Molecular Dimensions:* Using the MD11-51-100 plastic bases combined with the MD11-54 covers results in a cost of $9.20 per plate. Laminex Plastic 100 um base (MD11-51-100) - I've used these to solve multiple structures during my post-doc at NIH. They're UV transparent for doing UV microscopy and detection of your crystals. Cost: $66 USD for a pack of 10. Laminex Plastic 200 um base (MD11-50) - Has a thicker 200 um layer for inputting larger amounts of mesophase and precipitant. Cost: $66 USD for a pack of 10. Laminex Film Cover (MD11-54) - Used to cover the top of the sandwich plate. Also UV transparent. Cost: $26 USD per pack of 10. Laminex Glass 100 um base (MD11-50-100) - Glass base with 100 um spacer. Cost: $66 USD per pack of 10. Laminex Glass 200 um base (MD11-50) - Glass base with 200 um spacer. Cost: $66 USD per pack of 10. Glass Covers (MD11-52) - Glass for forming sandwich plates. Cost: $40 per pack of 10. *Hampton Research:* Plastic UV Transparent LCP Setup Kit (HR3-186) - Set of 20 plastic plates in a slightly different form factor than the Mol Dim. plastic plates. Cost: $483 USD per pack of 20. This is $24.15 per plate. Glass UV Transparent LCP Setup Kit (HR3-151) - Siliconized glass bases and glass tops. Cost: $368 USD per pack of 20 ($18.40 per plate). On Sat, Feb 25, 2012 at 12:35 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] Membrane Protein Crystallization Plates
QuoteParticulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) Since the person who asked this question here forget about it alltogether to write something back here is what he was asking about (i think) Anybody heard about the nextal plates see the link http://www.qiagen.com/products/nextalcubicphaseproducts.aspx It uses a different method of mixing lipids other than using the syringe method. They have lipid layered on the bottom of the well and protein samples are pipetted onto the monoolein.and later on precipitant. Plastic based ones are really bad idea Not everyone have an access to UV even if UV screening is used not a full-proof method to detect membrane protein micro crystals I would definitely think glass based plates are the way to go. On Sat, Feb 25, 2012 at 3:35 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] Desalting columns
Hi Sangeetha: If you just want to check which buffer is good for your protein, maybe you can try to set up a crystallization screen, keeping your protein concentration just 3 mg/ml. You can observe (after several days) which conditions give you a clear drop, and maybe you can find a clue which buffer is better for your protein. If you want to speed up the whole processes, you can also add glycerol into the drops to grasp the water molecules. Yu Xiaodi Date: Mon, 27 Feb 2012 11:01:33 -0500 From: sangeetha...@gmail.com Subject: [ccp4bb] Desalting columns To: CCP4BB@JISCMAIL.AC.UK Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
We did some data mining from remark 280 of the PDB in 2004. Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml. See table 3 at http://www.douglas.co.uk/PDB_data.htm . Patrick On 27 February 2012 16:25, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? ** ** For crystallization, the concentration needs to be **a) **high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) **b) **high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. ** ** There is ample evidence for proteins crystallizing below 3 mg/ml. ** ** The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. ** ** Sometimes the shape of a distribution matters ;-) ** ** BR ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Sangeetha Vedula *Sent:* Monday, February 27, 2012 8:02 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Desalting columns ** ** Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha. -- 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] Desalting columns
Hi, We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer. Protein-to-well-solution ratio was 3:1. When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio. That is another important variable to test, I guess. Regards, -Andre. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Monday, February 27, 2012 1:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
Ø in 2004. Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml. That is not necessarily the success rate for low concentrations, which we actually would like to have. We would need negatives for 3 for to give a correct answer. I guess even occurrence might be more frequent now. hintSomething for our center data miners who have the negatives kept /hint. Anecdotally, my personal below-3-occurance brag not success, that is 100% /brag is ~15%, but that does not mean much. Even if it were the success rate: 5% chance compared to zero chance when precipitated Id take it and probably learn something useful during the experiment. BR
Re: [ccp4bb] Membrane Protein Crystallization Plates
Thanks for all the replies. I will try a couple of different plates/set-ups. My favorite will be the one that gives me a crystal ;)
