[ccp4bb] Postdoctoral position in Michael Rossmann's laboratory
A post-doctoral position is available (in the department of Biological Sciences, Purdue University, Indiana 47907, USA) for structural studies of viruses and their interaction with host cells. Applicants should have experience in virus or protein crystallography and in molecular biology. Experience in electron microscopy would also be useful. Please send applications to Michael Rossmann ([EMAIL PROTECTED]).
Re: [ccp4bb] Helix builder
Not quite sure if I understand your question correctly. But how about Moleman from the USF suite ? Use the command HELIx_generate, then later mutate that helix into your sequence. Jürgen On 19 Sep 2008, at 11:19, john peter wrote: Hi CCP4ers, Apologies for this off-topic question, but I believe someone in this community could help me. Suppose if I know the position of every 3rd or 4th or n-th unit (amino acid or nucleotide or any moiety) of any helix (not just the protein & nucleic acid helices), is it possible to generate the complete the helix. If one could, could someone point out a software that could do it. Thanks in advance. Cheers John - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] Unexplained electron density
Dear Maria -- On 19 Sep 2008, at 01:38, Maria Håkansson wrote: Any suggestions? In addition, could that be a water molecule present at (or near) a special position? Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] off-topic: selective reduction of surface cysteine
We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation), Did you inhibit S-S bond formation after addition of SDS? Lots of proteins form dimers when unfolded without presence of reducing agent. Adding 20 mM NEM into SDS-PAGE loading buffer is easiest way to prevent this. Assuming that you did this and you actually see intermolecular bond, you can play with reducing agent(s) concentrations. Intramolecular bonds require much higher concentration of reducing agents (e.g., IgG is perfectly happy and not reduced at 1 mM DTT at room temperature). Dima
[ccp4bb] off-topic: selective reduction of surface cysteine
Dear all, We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation), and we also see other higher molecular weight species that are immunoreactive with an antibody to our protein, so maybe we have heterodimerization with other cysteine-containing proteins as well. Can anyone point me to references or protocols that might help us to selectively reduce our protein at the engineered Cys, breaking up the dimers, while preserving the disulfide structure of the native protein? Or might there be a way to reversibly protect the engineered Cys throughout expression and purification to prevent dimerization? Any suggestions are welcome. Thanks! Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Non-sequential residue numbering?
I would suggest depositors take a look at the PDB Exchange Dictionary and at the following definitions: _atom_site.auth_seq_id An alternative identifier for _atom_site.label_seq_id that may be provided by an author in order to match the identification used in the publication that describes the structure. Note that this is not necessarily a number, that the values do not have to be positive, and that the value does not have to correspond to the value of _atom_site.label_seq_id. The value of _atom_site.label_seq_id is required to be a sequential list of positive integers. The author may assign values to _atom_site.auth_seq_id in any desired way. For instance, the values may be used to relate this structure to a numbering scheme in a homologous structure, including sequence gaps or insertion codes. Alternatively, a scheme may be used for a truncated polymer that maintains the numbering scheme of the full length polymer. In all cases, the scheme used here must match the scheme used in the publication that describes the structure. _atom_site.label_seq_id This data item is a pointer to _entity_poly_seq.num in the ENTITY_POLY_SEQ category. _entity_poly_seq.num The value of _entity_poly_seq.num must uniquely and sequentially identify a record in the ENTITY_POLY_SEQ list. Note that this item must be a number and that the sequence numbers must progress in increasing numerical order. So, at the very least, the PDB's internal database and mmCIF and PDBML files should be able to handle _both_ the simplified numbering the annotator wishes to impose, and the more scientifically useful notation an author might use to place their structure in context. It should be a "simple" matter of programming for the PDB to produce "PDB" entries done either way. One should also note the the entire system of insertion codes does not make much sense without the broader contextual view of families of structures. Regards, Herbert At 2:33 PM -0400 9/19/08, Frances C. Bernstein wrote: I was at the PDB from 1974 - 1998 and closely involved with processing entries 15 to ~9000. We also designed the "PDB format". My replies were based on what was done for those 24 years and I cannot address what is currently being done at the PDB. I do not know if the current PDB staff follows this bulletin board and I can only suggest that you take this matter up with the current PDB management, the community, and the PDB advisory board. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 19 Sep 2008, Linda Brinen wrote: I'm actually pleased to read your response and interpretation of what is allowable and why, Frances. However, it's it pretty stark contrast to what I was told about 18 months ago when I struggled (and eventually lost) to preserve a numbering scheme that had a long standing historical and literature precedence when submitting a new structure to the PDB. This was a two-domain protein; the first domain - according to historical numbering - had a number plus a letter code to indicate the domain; the second domain, which started again with the number 1 - had no letter code. We were told that that was not allowed. We wanted to preserve insertions and deletions as well, but were also strongly discouraged, if not flat out told we could not. While it's not usually prudent to quote offline e-mail exchanges, I'm going to snip pertinent pieces of the discussion (I'm leaving the original spelling errors and text bolding in place) with no indication of the annotator who wrote these guidelines to our group. Here's part of one of the many 'exchanges' that was had: "I understand your point and that certain close research communities have certain habits and traditions but the PDB serves to the whole community of structural biology, bioinformatics, to many educators, students... In all these cases, the simplest possible numbering of sequences, ideally numbering identical to the numbering used by the UNP sequence database, is far the most useful because easiest to understand. I do not say this because it is in our manuals and help pages but because I have eight years of experience with annotation of all kinds of structures. I would therefore very much like to ask you to reconsider the way how you number your protein, yo
Re: [ccp4bb] Non-sequential residue numbering?
I was at the PDB from 1974 - 1998 and closely involved with processing entries 15 to ~9000. We also designed the "PDB format". My replies were based on what was done for those 24 years and I cannot address what is currently being done at the PDB. I do not know if the current PDB staff follows this bulletin board and I can only suggest that you take this matter up with the current PDB management, the community, and the PDB advisory board. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 19 Sep 2008, Linda Brinen wrote: I'm actually pleased to read your response and interpretation of what is allowable and why, Frances. However, it's it pretty stark contrast to what I was told about 18 months ago when I struggled (and eventually lost) to preserve a numbering scheme that had a long standing historical and literature precedence when submitting a new structure to the PDB. This was a two-domain protein; the first domain - according to historical numbering - had a number plus a letter code to indicate the domain; the second domain, which started again with the number 1 - had no letter code. We were told that that was not allowed. We wanted to preserve insertions and deletions as well, but were also strongly discouraged, if not flat out told we could not. While it's not usually prudent to quote offline e-mail exchanges, I'm going to snip pertinent pieces of the discussion (I'm leaving the original spelling errors and text bolding in place) with no indication of the annotator who wrote these guidelines to our group. Here's part of one of the many 'exchanges' that was had: "I understand your point and that certain close research communities have certain habits and traditions but the PDB serves to the whole community of structural biology, bioinformatics, to many educators, students... In all these cases, the simplest possible numbering of sequences, ideally numbering identical to the numbering used by the UNP sequence database, is far the most useful because easiest to understand. I do not say this because it is in our manuals and help pages but because I have eight years of experience with annotation of all kinds of structures. I would therefore very much like to ask you to reconsider the way how you number your protein, your numbering schema is *interpretation* more than a mere labeling schema. Needles to say, no sequence numbering can satisfy this ambition...from my point of view, especially the jump from 96P back to 1 will cause a lot of confusion and misunderstandinglook at the problem from a standpoint of a general naturalist instead of an narrow protease community" This left us with a mandated 'start from 1 and number sequentially' format that did exactly the opposite of what you, Frances, correctly mention as important in any numbering scheme: preserve relationships with other proteins. We've had to resort to providing 'translation tables' that identify what people were expecting to see as numbers for active site residues which now have new and non-sensical numbering. Is it the end of the world? Of course not. But neither is it necessarily the best scientific or logical presentation. At the risk of inciting a ratheranimated...dialogue on this topic, what has your experience been with this kind of thing (i.e., were we just unlucky??) and do current practices make sense and serve the community?? -Linda Frances C. Bernstein wrote: All entries list atoms starting at the N-terminus (or 5') so connectivity goes in the order of the atoms in the file - obviously with the possibility of unconnected portions where the density is inadequate. The entire philosphy of allowing numbering other than 1 - N had to do with preserving relationships with other proteins. The most common use relates to having an initial sequence 1 - N and then a similar sequence from another species with insertions and/or gaps. People wanted to be able to talk about the active site (which was preserved) using the same residue numbers. Negative numbers came up with additions at the N-terminus. Offhand, I don't recall why descending numbers were used but I believe that there is at least one such entry. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On F
[ccp4bb] Helix builder
Hi CCP4ers, Apologies for this off-topic question, but I believe someone in this community could help me. Suppose if I know the position of every 3rd or 4th or n-th unit (amino acid or nucleotide or any moiety) of any helix (not just the protein & nucleic acid helices), is it possible to generate the complete the helix. If one could, could someone point out a software that could do it. Thanks in advance. Cheers John
Re: [ccp4bb] Non-sequential residue numbering?
All entries list atoms starting at the N-terminus (or 5') so connectivity goes in the order of the atoms in the file - obviously with the possibility of unconnected portions where the density is inadequate. The entire philosphy of allowing numbering other than 1 - N had to do with preserving relationships with other proteins. The most common use relates to having an initial sequence 1 - N and then a similar sequence from another species with insertions and/or gaps. People wanted to be able to talk about the active site (which was preserved) using the same residue numbers. Negative numbers came up with additions at the N-terminus. Offhand, I don't recall why descending numbers were used but I believe that there is at least one such entry. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 19 Sep 2008, Ian Tickle wrote: But what connectivity would be implied by descending numbers: the order in the file or the order of the numbering? I assume the former, otherwise what would be the point of having descending numbering? And I wonder how many programs would baulk at it (or even at ascending negative numbers?). -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Frances C. Bernstein Sent: 19 September 2008 16:44 To: Todd Geders Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Non-sequential residue numbering? As long as each residue within a chain has a unique identifier (residue number plus insertion code), there is no restriction on numbering. The numbers can be in ascending or descending order, non-sequential, and even negative. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 19 Sep 2008, Todd Geders wrote: Hello all, I have a structure from a non-natural fusion of the truncated C-terminus of one protein with the truncated N-terminus of another. For the deposition, we want to keep the numbering as found in the separate proteins. It looks something like this: 1 12 | | HWVCKDIALLMCFFLEEMSEEP || 754 763 At no point is there an overlap in numbering (i.e. the N-terminal residue number is higher than the C-terminal residue number). Is this numbering scheme supported by the PDB standard? Thus far, all of the software seems to handle it (refmac, Coot, PyMOL, pdb_extract, PDB precheck & validation, etc). Can anyone see a reason to not deposit with this non-sequential residue numbering? ~Todd Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Non-sequential residue numbering?
But what connectivity would be implied by descending numbers: the order in the file or the order of the numbering? I assume the former, otherwise what would be the point of having descending numbering? And I wonder how many programs would baulk at it (or even at ascending negative numbers?). -- Ian > -Original Message- > From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On > Behalf Of Frances C. Bernstein > Sent: 19 September 2008 16:44 > To: Todd Geders > Cc: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Non-sequential residue numbering? > > As long as each residue within a chain has a unique identifier > (residue number plus insertion code), there is no restriction > on numbering. The numbers can be in ascending or descending > order, non-sequential, and even negative. > > Frances > > = > Bernstein + Sons > * * Information Systems Consultants > 5 Brewster Lane, Bellport, NY 11713-2803 > * * *** > *Frances C. Bernstein >* *** [EMAIL PROTECTED] > *** * >* *** 1-631-286-1339FAX: 1-631-286-1999 > = > > On Fri, 19 Sep 2008, Todd Geders wrote: > > > Hello all, > > > > I have a structure from a non-natural fusion of the truncated C-terminus > of > > one protein with the truncated N-terminus of another. For the > deposition, we > > want to keep the numbering as found in the separate proteins. It looks > > something like this: > > > > 1 12 > > | | > > HWVCKDIALLMCFFLEEMSEEP > > || > > 754 763 > > > > At no point is there an overlap in numbering (i.e. the N-terminal > residue > > number is higher than the C-terminal residue number). > > > > Is this numbering scheme supported by the PDB standard? Thus far, all > of the > > software seems to handle it (refmac, Coot, PyMOL, pdb_extract, PDB > precheck & > > validation, etc). > > > > Can anyone see a reason to not deposit with this non-sequential residue > > numbering? > > > > ~Todd Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Non-sequential residue numbering?
As long as each residue within a chain has a unique identifier (residue number plus insertion code), there is no restriction on numbering. The numbers can be in ascending or descending order, non-sequential, and even negative. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** [EMAIL PROTECTED] *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 19 Sep 2008, Todd Geders wrote: Hello all, I have a structure from a non-natural fusion of the truncated C-terminus of one protein with the truncated N-terminus of another. For the deposition, we want to keep the numbering as found in the separate proteins. It looks something like this: 1 12 | | HWVCKDIALLMCFFLEEMSEEP || 754 763 At no point is there an overlap in numbering (i.e. the N-terminal residue number is higher than the C-terminal residue number). Is this numbering scheme supported by the PDB standard? Thus far, all of the software seems to handle it (refmac, Coot, PyMOL, pdb_extract, PDB precheck & validation, etc). Can anyone see a reason to not deposit with this non-sequential residue numbering? ~Todd
[ccp4bb] Non-sequential residue numbering?
Hello all, I have a structure from a non-natural fusion of the truncated C- terminus of one protein with the truncated N-terminus of another. For the deposition, we want to keep the numbering as found in the separate proteins. It looks something like this: 1 12 | | HWVCKDIALLMCFFLEEMSEEP || 754 763 At no point is there an overlap in numbering (i.e. the N-terminal residue number is higher than the C-terminal residue number). Is this numbering scheme supported by the PDB standard? Thus far, all of the software seems to handle it (refmac, Coot, PyMOL, pdb_extract, PDB precheck & validation, etc). Can anyone see a reason to not deposit with this non-sequential residue numbering? ~Todd
Re: [ccp4bb] Unexplained electron density
Maria, 1.7 A is too long for common diatomic molecules (N2, 1.1 A; O2, 1.2 A; CO, 1.1 A) or molecules like hydrogen peroxide (~1.48 A), methanol (ditto), etc. Does the "water" have unusually low temp. factors (i.e., it's really a much heavier atom, and the peak you see is a Fourier ripple)? It may be indeed that you have (one) water at two alternate positions, but I think you need to reset to nothing modeled to be certain of density interpretaion moving forward. Thus, try removing the water you have already built, any surrounding waters or other ligands, and also the protein atoms to which all these are hydrogen bonded (set occ = 0), refine a few cycles, and then look at the diff. maps. Dave David Borhani, Ph.D. D. E. Shaw Research, LLC 120 West Forty-Fifth Street, 39th Floor New York, NY 10036 [EMAIL PROTECTED] 212-478-0698 http://www.deshawresearch.com > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On > Behalf Of Maria Håkansson > Sent: Friday, September 19, 2008 8:00 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Unexplained electron density > > Hello David and others, > > Thanks for yur comments. > > I guess it might be as simple as water molecules, present in the > structure but not at the same time. > > The density looks like a rod with uneven distribution. Both ends of > the rods (1.7-1.8 Å in between) > make hydrogen bonds to protein or other water molecules - normal > distances (2.3-3.3 Å). Could it be > strong water molecules but with partial occupancy meaning that both > sites are occupied but not at the same time? > > I guess refmac automatically refines the molecules that way > although I > have not specified it in my file. So after > refinement as too close water molecules there is no clash just nice > density. However I assume it is appropriate to > specify these water molecules as the same water but with an > alternative conformation in the pdb file. > > Best Regards, > > Maria > > > > > 19 Sep 2008 kl. 11:10 skrev David Briggs: > > > Hi Maria, > > > > Initial questions: > > 1) What's present in crystallisation/purification buffers? > > 2) Are any other ligand visible for the 9sigma peak? > > 3) Does the 9 sigma peak also have a peak in an anomalous > difference > > map? > > > > Assuming 1.7A is accurate (and with 1.5A resolution, you'd hope it > > would be!) a metal-ion - water interaction is looking less likely. > > Take a look at Marjorie Harding's papers and website for > target metal > > ion - ligand distances, and the closest you'll see is water:Mg2+, at > > 2.07. > > > > http://tanna.bch.ed.ac.uk/newtargs_06.html > > > > So one would assume it is a covalently bonded compound. > > So back to question 1. > > > > What's in your buffers? > > A quick search for bond length tables suggests Carbon-Sulphur (1.8) > > and Carbon-Chlorine at 1.7. > > > > hope this helps > > > > David > > > > 2008/9/19 Maria Håkansson <[EMAIL PROTECTED]>: > >> Hello All, > >> > >> I have a problem with a 9 sigma positive peak 1.7 Å away > from a water > >> molecule (or what I believe is > >> a water molecule). There are several similar peaks in my > map though > >> only one > >> is as high as 9 sigma. > >> > >> My first thought was to exclude these too close waters. > However the > >> R-values > >> increased by more than 0.5 %. Could it > >> be carbonmonoxide or oxygen atoms? By the way, It is 1.5 Å > >> resolution data. > >> Any suggestions? > >> > >> Best Regards, > >> > >> Maria Håkansson > >> > >> > >>> Maria Håkansson, Ph.D. > >>> tel: +46 (0) 76 8585 706 > >>> Senior Scientist, Max-lab, Lund University > >>> fax: +46 46 222 47 10 > >>> Ole Römers väg 1 (P.O. Box 188) > >>> www.maxlab.lu.se > >>> SE-221 00 Lund, Sweden > [EMAIL PROTECTED] > >>> > >> > > > > > > > > -- > > > > David C. Briggs PhD > > Father & Crystallographer > > http://drdavidcbriggs.googlepages.com/home > > AIM ID: dbassophile > > > > > Maria Håkansson, Ph.D. > > tel: +46 (0) 76 8585 706 > > Senior Scientist, SARomics Biostructures ABfax: +46 46 19 12 77 > > Scheelevägen 22 (P.O. Box 724) > www.saromicsbiostructures.com > > SE-220 07 Lund, Sweden > [EMAIL PROTECTED] > > >
Re: [ccp4bb] Unexplained electron density
Hello David and others, Thanks for yur comments. I guess it might be as simple as water molecules, present in the structure but not at the same time. The density looks like a rod with uneven distribution. Both ends of the rods (1.7-1.8 Å in between) make hydrogen bonds to protein or other water molecules - normal distances (2.3-3.3 Å). Could it be strong water molecules but with partial occupancy meaning that both sites are occupied but not at the same time? I guess refmac automatically refines the molecules that way although I have not specified it in my file. So after refinement as too close water molecules there is no clash just nice density. However I assume it is appropriate to specify these water molecules as the same water but with an alternative conformation in the pdb file. Best Regards, Maria 19 Sep 2008 kl. 11:10 skrev David Briggs: Hi Maria, Initial questions: 1) What's present in crystallisation/purification buffers? 2) Are any other ligand visible for the 9sigma peak? 3) Does the 9 sigma peak also have a peak in an anomalous difference map? Assuming 1.7A is accurate (and with 1.5A resolution, you'd hope it would be!) a metal-ion - water interaction is looking less likely. Take a look at Marjorie Harding's papers and website for target metal ion - ligand distances, and the closest you'll see is water:Mg2+, at 2.07. http://tanna.bch.ed.ac.uk/newtargs_06.html So one would assume it is a covalently bonded compound. So back to question 1. What's in your buffers? A quick search for bond length tables suggests Carbon-Sulphur (1.8) and Carbon-Chlorine at 1.7. hope this helps David 2008/9/19 Maria Håkansson <[EMAIL PROTECTED]>: Hello All, I have a problem with a 9 sigma positive peak 1.7 Å away from a water molecule (or what I believe is a water molecule). There are several similar peaks in my map though only one is as high as 9 sigma. My first thought was to exclude these too close waters. However the R-values increased by more than 0.5 %. Could it be carbonmonoxide or oxygen atoms? By the way, It is 1.5 Å resolution data. Any suggestions? Best Regards, Maria Håkansson Maria Håkansson, Ph.D. tel: +46 (0) 76 8585 706 Senior Scientist, Max-lab, Lund University fax: +46 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) www.maxlab.lu.se SE-221 00 Lund, Sweden [EMAIL PROTECTED] -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home AIM ID: dbassophile Maria Håkansson, Ph.D. tel: +46 (0) 76 8585 706 Senior Scientist, SARomics Biostructures ABfax: +46 46 19 12 77 Scheelevägen 22 (P.O. Box 724) www.saromicsbiostructures.com SE-220 07 Lund, Sweden [EMAIL PROTECTED]
Re: [ccp4bb] Unexplained electron density
Hi Maria, Initial questions: 1) What's present in crystallisation/purification buffers? 2) Are any other ligand visible for the 9sigma peak? 3) Does the 9 sigma peak also have a peak in an anomalous difference map? Assuming 1.7A is accurate (and with 1.5A resolution, you'd hope it would be!) a metal-ion - water interaction is looking less likely. Take a look at Marjorie Harding's papers and website for target metal ion - ligand distances, and the closest you'll see is water:Mg2+, at 2.07. http://tanna.bch.ed.ac.uk/newtargs_06.html So one would assume it is a covalently bonded compound. So back to question 1. What's in your buffers? A quick search for bond length tables suggests Carbon-Sulphur (1.8) and Carbon-Chlorine at 1.7. hope this helps David 2008/9/19 Maria Håkansson <[EMAIL PROTECTED]>: > Hello All, > > I have a problem with a 9 sigma positive peak 1.7 Å away from a water > molecule (or what I believe is > a water molecule). There are several similar peaks in my map though only one > is as high as 9 sigma. > > My first thought was to exclude these too close waters. However the R-values > increased by more than 0.5 %. Could it > be carbonmonoxide or oxygen atoms? By the way, It is 1.5 Å resolution data. > Any suggestions? > > Best Regards, > > Maria Håkansson > > >> Maria Håkansson, Ph.D. >> tel: +46 (0) 76 8585 706 >> Senior Scientist, Max-lab, Lund University >> fax: +46 46 222 47 10 >> Ole Römers väg 1 (P.O. Box 188) >> www.maxlab.lu.se >> SE-221 00 Lund, Sweden [EMAIL PROTECTED] >> > -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home AIM ID: dbassophile
