Re: [ccp4bb] Bug in c_truncate? - phase mods
CAD and Kevins phasematch correctly change phases etc when you change symmetry operator. I cant think that this is the job for pointless.. it is responsible for intensities, and surely only needs to use a merged file to decide on the appropriate choice of axes - eg getting your new PG3 data set on the same indexing convention? Why would you need to modify the reference data? Eleanor On 11/01/2010 03:41 PM, Ian Tickle wrote: Dealing with the phases (and therefore also the Hendrickson-Lattman coefficients) on re-indexing is trivial: the phases are not changed by re-indexing because the inverse transformation must simultaneously be applied to the co-ordinates. This is because in general the re-indexing transformation is not necessarily a symmetry operator (think of P1), so you can't rely on being able to compensate for the effect on the co-ordinates by using a symmetry operator. So the effect on the phases cancels out ... unless of course your re-indexing operator inverts the hand, in which case you almost certainly don't want also to invert the hand of your co-ordinates, so in that case you must compensate by transforming the structure factors to their complex conjugates (i.e. multiply phases by -1). I guess you're thinking of the subsequent necessary transformation of the indices to the asymmetric unit, where the phases& H-L coeffs do in general change (because then you are only changing the indices, not the co-ordinates); however CAD will do that transformation for you. Incidentally this is a neat illustration of the difference between a vector and a complex number. The re-indexing transformation is a transformation of the reference frame, which as long as it doesn't invert the hand, leaves the complex structure factors invariant, so they must be complex scalars (except in centric zones where they can sometimes be represented by real scalars). The indices (whether reflection or Miller!) obviously form a 3-D vector with integer elements (unless of course you're interested in diffuse scattering when they have to be reals). Either way, this is a vector because in the general case (there will be exceptions for reflections on symmetry axes) its elements change on re-indexing (that's what re-indexing means!). If the structure were in 1-D or 2-D exactly the same would apply: the 1- or 2-D elements would still in general change on transforming the reference frame so would be represented by 1- or 2-D vectors; the structure factors would still be invariant, thus illustrating the important difference between a real scalar and a 1-D vector, and between a complex scalar and a 2-D vector. Cheers --Ian On Mon, Nov 1, 2010 at 1:28 PM, Phil Evans wrote: I can see we need to make sure that data can come in at any point, as Is of Fs Pointless can do automatic reindexing to a reference, and will preserve all columns from a merged file, but can't cope with phases, as I've not got round to working out appropriate phase shifts on reindexing Phil On 1 Nov 2010, at 13:17, Ian Tickle wrote: Phil Yes our processing pipeline absolutely has to be able to take in data from any internal (in-house X-ray or synchrotron) or external (PDB or collaborator's) source, including those where I, sigI and freeR flag are present. One of the first things I did was to modify truncate so it would pass through the freeR flag column. If the I/sigI are present I always strip out the F/sigF columns. So it seemed logical to run truncate as the very last step, e.g.: 1. sortmtz 2. scala Steps 1& 2 only for internally collected or unmerged data. 3. refindexExternal merged data enters pipeline here: auto-re-index to reference. 4. cad Sort; put into standard a.u.; add freeR column from reference if not already present. 5. rescut My own prog for auto-determination of resolution cutoff based on shell & completeness. 6. truncate Apply resolution cutoff; if Is available convert to Fs. I always run steps 3-6 in that order. I always check that the resolution cutoff is sensible& if Is are available I always run truncate to ensure it's done properly (i.e. correct cell contents are specified). I'm still using truncate because AFAICS ctruncate couldn't handle freeR flags (maybe that's fixed now, maybe not). Also truncate produces a more informative N(Z) plot which shows the expected distribution for a twinned crystal (I believe this feature has now been added to ctruncate). Cheers -- Ian On Fri, Oct 29, 2010 at 1:05 PM, Phil Evans wrote: The normal use of [c]truncate is to take intensities from Scala, so it wouldn't expect FreeR flags in the file. I suppose this should be added for other uses of the program Is this something that is often used? Do people import intensities into CCP4 to convert them to Fs? Phil On 29 Oct 2010, at 13:01, herman.schreu...@sanofi-aventis.com wrote: Dear Peter, Since I did not hear that your problem is solved here my two cents. I did some tests using the
Re: [ccp4bb] Bug in c_truncate? - phase mods
Re-indexing of an input (either merged or unmerged) dataset to a (also merged or unmerged) reference dataset is one of the 'subsidiary' functions of pointless, according to Phil's man page: This mode is selected if an HKLREF dataset is specified. Given a test dataset, merged or unmerged (file(s) HKLIN), and a merged or unmerged reference dataset in a known space group (file HKLREF), the program tests all possible alternative indexing schemes of the test dataset to find which one best matches the reference set. Not sure what you mean by "Why would you need to modify the reference data?" ? The reference data is not modified, who said it was? -- Ian On Tue, Nov 2, 2010 at 7:03 AM, Eleanor Dodson wrote: > CAD and Kevins phasematch correctly change phases etc when you change > symmetry operator. > > I cant think that this is the job for pointless.. it is responsible for > intensities, and surely only needs to use a merged file to decide on the > appropriate choice of axes - eg getting your new PG3 data set on the same > indexing convention? Why would you need to modify the reference data? > Eleanor > > On 11/01/2010 03:41 PM, Ian Tickle wrote: >> >> Dealing with the phases (and therefore also the Hendrickson-Lattman >> coefficients) on re-indexing is trivial: the phases are not changed by >> re-indexing because the inverse transformation must simultaneously be >> applied to the co-ordinates. This is because in general the >> re-indexing transformation is not necessarily a symmetry operator >> (think of P1), so you can't rely on being able to compensate for the >> effect on the co-ordinates by using a symmetry operator. So the >> effect on the phases cancels out ... unless of course your >> re-indexing operator inverts the hand, in which case you almost >> certainly don't want also to invert the hand of your co-ordinates, so >> in that case you must compensate by transforming the structure factors >> to their complex conjugates (i.e. multiply phases by -1). I guess >> you're thinking of the subsequent necessary transformation of the >> indices to the asymmetric unit, where the phases& H-L coeffs do in >> general change (because then you are only changing the indices, not >> the co-ordinates); however CAD will do that transformation for you. >> >> Incidentally this is a neat illustration of the difference between a >> vector and a complex number. The re-indexing transformation is a >> transformation of the reference frame, which as long as it doesn't >> invert the hand, leaves the complex structure factors invariant, so >> they must be complex scalars (except in centric zones where they can >> sometimes be represented by real scalars). The indices (whether >> reflection or Miller!) obviously form a 3-D vector with integer >> elements (unless of course you're interested in diffuse scattering >> when they have to be reals). Either way, this is a vector because in >> the general case (there will be exceptions for reflections on symmetry >> axes) its elements change on re-indexing (that's what re-indexing >> means!). If the structure were in 1-D or 2-D exactly the same would >> apply: the 1- or 2-D elements would still in general change on >> transforming the reference frame so would be represented by 1- or 2-D >> vectors; the structure factors would still be invariant, thus >> illustrating the important difference between a real scalar and a 1-D >> vector, and between a complex scalar and a 2-D vector. >> >> Cheers >> >> --Ian >> >> On Mon, Nov 1, 2010 at 1:28 PM, Phil Evans wrote: >>> >>> I can see we need to make sure that data can come in at any point, as Is >>> of Fs >>> >>> Pointless can do automatic reindexing to a reference, and will preserve >>> all columns from a merged file, but can't cope with phases, as I've not got >>> round to working out appropriate phase shifts on reindexing >>> >>> Phil >>> >>> >>> On 1 Nov 2010, at 13:17, Ian Tickle wrote: >>> Phil Yes our processing pipeline absolutely has to be able to take in data from any internal (in-house X-ray or synchrotron) or external (PDB or collaborator's) source, including those where I, sigI and freeR flag are present. One of the first things I did was to modify truncate so it would pass through the freeR flag column. If the I/sigI are present I always strip out the F/sigF columns. So it seemed logical to run truncate as the very last step, e.g.: 1. sortmtz 2. scala Steps 1& 2 only for internally collected or unmerged data. 3. refindex External merged data enters pipeline here: auto-re-index to reference. 4. cad Sort; put into standard a.u.; add freeR column from reference if not already present. 5. rescut My own prog for auto-determination of resolution cutoff based on shell & completeness. 6. truncate Apply resolution cutoff; if Is available convert to Fs. I always run steps 3-6
Re: [ccp4bb] Bug in c_truncate? - phase mods
As Ian says, it doesn't modify the reference set. Yes it is a subsidiary function of Pointless, aimed at replacing the program REINDEX (since much of the functionality was already in there, it seemed sensible to add this option). It ought to do appropriate phase changes, but like Reindex, it doesn't at present. My feeling was that you can always recalculate the phases after a reindexing. Phil On 2 Nov 2010, at 08:28, Ian Tickle wrote: > Re-indexing of an input (either merged or unmerged) dataset to a (also > merged or unmerged) reference dataset is one of the 'subsidiary' > functions of pointless, according to Phil's man page: > > This mode is selected if an HKLREF dataset is specified. > Given a test dataset, merged or unmerged (file(s) HKLIN), and a merged > or unmerged reference dataset in a known space group (file HKLREF), the > program tests all possible alternative indexing schemes of the test > dataset to find which one best matches the reference set. > > Not sure what you mean by "Why would you need to modify the reference > data?" ? The reference data is not modified, who said it was? > > -- Ian > > On Tue, Nov 2, 2010 at 7:03 AM, Eleanor Dodson wrote: >> CAD and Kevins phasematch correctly change phases etc when you change >> symmetry operator. >> >> I cant think that this is the job for pointless.. it is responsible for >> intensities, and surely only needs to use a merged file to decide on the >> appropriate choice of axes - eg getting your new PG3 data set on the same >> indexing convention? Why would you need to modify the reference data? >> Eleanor >> >> On 11/01/2010 03:41 PM, Ian Tickle wrote: >>> >>> Dealing with the phases (and therefore also the Hendrickson-Lattman >>> coefficients) on re-indexing is trivial: the phases are not changed by >>> re-indexing because the inverse transformation must simultaneously be >>> applied to the co-ordinates. This is because in general the >>> re-indexing transformation is not necessarily a symmetry operator >>> (think of P1), so you can't rely on being able to compensate for the >>> effect on the co-ordinates by using a symmetry operator. So the >>> effect on the phases cancels out ... unless of course your >>> re-indexing operator inverts the hand, in which case you almost >>> certainly don't want also to invert the hand of your co-ordinates, so >>> in that case you must compensate by transforming the structure factors >>> to their complex conjugates (i.e. multiply phases by -1). I guess >>> you're thinking of the subsequent necessary transformation of the >>> indices to the asymmetric unit, where the phases& H-L coeffs do in >>> general change (because then you are only changing the indices, not >>> the co-ordinates); however CAD will do that transformation for you. >>> >>> Incidentally this is a neat illustration of the difference between a >>> vector and a complex number. The re-indexing transformation is a >>> transformation of the reference frame, which as long as it doesn't >>> invert the hand, leaves the complex structure factors invariant, so >>> they must be complex scalars (except in centric zones where they can >>> sometimes be represented by real scalars). The indices (whether >>> reflection or Miller!) obviously form a 3-D vector with integer >>> elements (unless of course you're interested in diffuse scattering >>> when they have to be reals). Either way, this is a vector because in >>> the general case (there will be exceptions for reflections on symmetry >>> axes) its elements change on re-indexing (that's what re-indexing >>> means!). If the structure were in 1-D or 2-D exactly the same would >>> apply: the 1- or 2-D elements would still in general change on >>> transforming the reference frame so would be represented by 1- or 2-D >>> vectors; the structure factors would still be invariant, thus >>> illustrating the important difference between a real scalar and a 1-D >>> vector, and between a complex scalar and a 2-D vector. >>> >>> Cheers >>> >>> --Ian >>> >>> On Mon, Nov 1, 2010 at 1:28 PM, Phil Evans wrote: I can see we need to make sure that data can come in at any point, as Is of Fs Pointless can do automatic reindexing to a reference, and will preserve all columns from a merged file, but can't cope with phases, as I've not got round to working out appropriate phase shifts on reindexing Phil On 1 Nov 2010, at 13:17, Ian Tickle wrote: > Phil > > Yes our processing pipeline absolutely has to be able to take in data > from any internal (in-house X-ray or synchrotron) or external (PDB or > collaborator's) source, including those where I, sigI and freeR flag > are present. One of the first things I did was to modify truncate so > it would pass through the freeR flag column. If the I/sigI are > present I always strip out the F/sigF columns. So it seemed logical > to run truncate as
Re: [ccp4bb] Bug in c_truncate? - phase mods
I would say that being able to take in phases & H-L coeffs would be a useful option, because sometimes (I accept it's not mandatory) phases are available in SF files from PDB which allows us to see exactly the same map that was interpreted by the depositor. In some cases there's some doubt that the density for an inhibitor or whatever claimed by the depositor is actually there (usually of course that's the whole point of their paper!!!). If you recalculate the phases there's always a nagging doubt that you've done something to change the density (e.g. different solvent model or whatever). The depositor's own density is damning evidence! Cheers -- Ian On Tue, Nov 2, 2010 at 8:56 AM, Phil Evans wrote: > As Ian says, it doesn't modify the reference set. > > Yes it is a subsidiary function of Pointless, aimed at replacing the program > REINDEX (since much of the functionality was already in there, it seemed > sensible to add this option). It ought to do appropriate phase changes, but > like Reindex, it doesn't at present. My feeling was that you can always > recalculate the phases after a reindexing. > > Phil > > On 2 Nov 2010, at 08:28, Ian Tickle wrote: > >> Re-indexing of an input (either merged or unmerged) dataset to a (also >> merged or unmerged) reference dataset is one of the 'subsidiary' >> functions of pointless, according to Phil's man page: >> >> This mode is selected if an HKLREF dataset is specified. >> Given a test dataset, merged or unmerged (file(s) HKLIN), and a merged >> or unmerged reference dataset in a known space group (file HKLREF), the >> program tests all possible alternative indexing schemes of the test >> dataset to find which one best matches the reference set. >> >> Not sure what you mean by "Why would you need to modify the reference >> data?" ? The reference data is not modified, who said it was? >> >> -- Ian >> >> On Tue, Nov 2, 2010 at 7:03 AM, Eleanor Dodson wrote: >>> CAD and Kevins phasematch correctly change phases etc when you change >>> symmetry operator. >>> >>> I cant think that this is the job for pointless.. it is responsible for >>> intensities, and surely only needs to use a merged file to decide on the >>> appropriate choice of axes - eg getting your new PG3 data set on the same >>> indexing convention? Why would you need to modify the reference data? >>> Eleanor >>> >>> On 11/01/2010 03:41 PM, Ian Tickle wrote: Dealing with the phases (and therefore also the Hendrickson-Lattman coefficients) on re-indexing is trivial: the phases are not changed by re-indexing because the inverse transformation must simultaneously be applied to the co-ordinates. This is because in general the re-indexing transformation is not necessarily a symmetry operator (think of P1), so you can't rely on being able to compensate for the effect on the co-ordinates by using a symmetry operator. So the effect on the phases cancels out ... unless of course your re-indexing operator inverts the hand, in which case you almost certainly don't want also to invert the hand of your co-ordinates, so in that case you must compensate by transforming the structure factors to their complex conjugates (i.e. multiply phases by -1). I guess you're thinking of the subsequent necessary transformation of the indices to the asymmetric unit, where the phases& H-L coeffs do in general change (because then you are only changing the indices, not the co-ordinates); however CAD will do that transformation for you. Incidentally this is a neat illustration of the difference between a vector and a complex number. The re-indexing transformation is a transformation of the reference frame, which as long as it doesn't invert the hand, leaves the complex structure factors invariant, so they must be complex scalars (except in centric zones where they can sometimes be represented by real scalars). The indices (whether reflection or Miller!) obviously form a 3-D vector with integer elements (unless of course you're interested in diffuse scattering when they have to be reals). Either way, this is a vector because in the general case (there will be exceptions for reflections on symmetry axes) its elements change on re-indexing (that's what re-indexing means!). If the structure were in 1-D or 2-D exactly the same would apply: the 1- or 2-D elements would still in general change on transforming the reference frame so would be represented by 1- or 2-D vectors; the structure factors would still be invariant, thus illustrating the important difference between a real scalar and a 1-D vector, and between a complex scalar and a 2-D vector. Cheers --Ian On Mon, Nov 1, 2010 at 1:28 PM, Phil Evans wrote: > > I can see we need to make sure that data can come in at any point, as Is > of Fs > >>
Re: [ccp4bb] Bug in c_truncate? - phase mods
I'll add it at some stage Phil On 2 Nov 2010, at 09:38, Ian Tickle wrote: > I would say that being able to take in phases & H-L coeffs would be a > useful option, because sometimes (I accept it's not mandatory) phases > are available in SF files from PDB which allows us to see exactly the > same map that was interpreted by the depositor. In some cases there's > some doubt that the density for an inhibitor or whatever claimed by > the depositor is actually there (usually of course that's the whole > point of their paper!!!). If you recalculate the phases there's > always a nagging doubt that you've done something to change the > density (e.g. different solvent model or whatever). The depositor's > own density is damning evidence! > > Cheers > > -- Ian > > On Tue, Nov 2, 2010 at 8:56 AM, Phil Evans wrote: >> As Ian says, it doesn't modify the reference set. >> >> Yes it is a subsidiary function of Pointless, aimed at replacing the program >> REINDEX (since much of the functionality was already in there, it seemed >> sensible to add this option). It ought to do appropriate phase changes, but >> like Reindex, it doesn't at present. My feeling was that you can always >> recalculate the phases after a reindexing. >> >> Phil >> >> On 2 Nov 2010, at 08:28, Ian Tickle wrote: >> >>> Re-indexing of an input (either merged or unmerged) dataset to a (also >>> merged or unmerged) reference dataset is one of the 'subsidiary' >>> functions of pointless, according to Phil's man page: >>> >>> This mode is selected if an HKLREF dataset is specified. >>> Given a test dataset, merged or unmerged (file(s) HKLIN), and a merged >>> or unmerged reference dataset in a known space group (file HKLREF), the >>> program tests all possible alternative indexing schemes of the test >>> dataset to find which one best matches the reference set. >>> >>> Not sure what you mean by "Why would you need to modify the reference >>> data?" ? The reference data is not modified, who said it was? >>> >>> -- Ian >>> >>> On Tue, Nov 2, 2010 at 7:03 AM, Eleanor Dodson wrote: CAD and Kevins phasematch correctly change phases etc when you change symmetry operator. I cant think that this is the job for pointless.. it is responsible for intensities, and surely only needs to use a merged file to decide on the appropriate choice of axes - eg getting your new PG3 data set on the same indexing convention? Why would you need to modify the reference data? Eleanor On 11/01/2010 03:41 PM, Ian Tickle wrote: > > Dealing with the phases (and therefore also the Hendrickson-Lattman > coefficients) on re-indexing is trivial: the phases are not changed by > re-indexing because the inverse transformation must simultaneously be > applied to the co-ordinates. This is because in general the > re-indexing transformation is not necessarily a symmetry operator > (think of P1), so you can't rely on being able to compensate for the > effect on the co-ordinates by using a symmetry operator. So the > effect on the phases cancels out ... unless of course your > re-indexing operator inverts the hand, in which case you almost > certainly don't want also to invert the hand of your co-ordinates, so > in that case you must compensate by transforming the structure factors > to their complex conjugates (i.e. multiply phases by -1). I guess > you're thinking of the subsequent necessary transformation of the > indices to the asymmetric unit, where the phases& H-L coeffs do in > general change (because then you are only changing the indices, not > the co-ordinates); however CAD will do that transformation for you. > > Incidentally this is a neat illustration of the difference between a > vector and a complex number. The re-indexing transformation is a > transformation of the reference frame, which as long as it doesn't > invert the hand, leaves the complex structure factors invariant, so > they must be complex scalars (except in centric zones where they can > sometimes be represented by real scalars). The indices (whether > reflection or Miller!) obviously form a 3-D vector with integer > elements (unless of course you're interested in diffuse scattering > when they have to be reals). Either way, this is a vector because in > the general case (there will be exceptions for reflections on symmetry > axes) its elements change on re-indexing (that's what re-indexing > means!). If the structure were in 1-D or 2-D exactly the same would > apply: the 1- or 2-D elements would still in general change on > transforming the reference frame so would be represented by 1- or 2-D > vectors; the structure factors would still be invariant, thus > illustrating the important difference between a real scalar and a 1-D > vector, and between a complex scalar and a 2-D vector. >
[ccp4bb] jligand
Dear All New version of jligand - version 1.0.0 is now available to download and use. It can be downloaded from http://www.ysbl.york.ac.uk/mxstat/ There are also tutorials how to jligand to create ligand and link descriptions. jligand is a program to create new ligand descriptions. It also can create description of covalent links between ligands as well as ligand proteins. These descriptions are used by refmac5 as well as coot (Paul will correct me if I am wrong) If you have any problems or suggestions please send an email to Andrey (preferable option) or me. With best regards Andrey and Garib
Re: [ccp4bb] Crystal gel band
It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
Re: [ccp4bb] Crystal gel band
The other thing you can try to do is "the-cheap-men-silver-stain" scan your gel and bump up the contrast, you'd be surprised what you can detect even from a regular stained gel. Best results are if you make the gel first as a greyscale image. Google for Neuhoff stain and bump up the phosphoric acid to 10%. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Nov 2, 2010, at 10:19 AM, Jim Pflugrath wrote: > It reads like you need to run a lane or two with a positive control of some > kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals > of a protein around the same expected molecular weight and try run on the gel > lanes with about the same amount of crystalline volume as your putative > protein crystals? > > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > xaravich ivan > Sent: Monday, November 01, 2010 9:51 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Crystal gel band > > Hi everyone, > I have grown some crystals after micro-seeding starting from thin-small > needles from needle-clusters. These crystals are larger in size than the > needles but are comparable to the shape and don't look like salt crystals. > But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a > home source,handy and would like to send these to the synchrotron. > > Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, > the amount of protein is < 1uG? > Has anyone experienced such a thing (no band in gel, but crystal diffracts)? > It would be nice if I get observations/suggestions. > > ivan
[ccp4bb] Software to Produce Linear Map of Surface Accessible Residues
Dear All, I'm looking for a software program to produce, given a 3D atomic structure of a molecule, a linear map showing the surface accessibility of residues in a protein structure. Would any one know of a program that can produce this sort of map. Thanks! and all the best, --Buz
Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues
Hello Buz, I do not know what you mean by 'linear map', but according to its manual, the ccp4-program "surface" writes a list of accessible are per atom per residue, which you could convert into the total fraction per residue with not too much effort. Is this what you are looking for? Cheers, Tim On Tue, Nov 02, 2010 at 10:36:56AM -0400, Buz Barstow wrote: > Dear All, > > I'm looking for a software program to produce, given a 3D atomic structure of > a molecule, a linear map showing the surface accessibility of residues in a > protein structure. > > Would any one know of a program that can produce this sort of map. > > Thanks! and all the best, > > --Buz -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues
Hi, As far as I know, thanks to Ian, areaimol in its latest versions now has an option of showing the fraction of accessible area per residue (relative to its accessible surface in the Gly-X-Gly peptide) both in the log file and in the B-factor column of the pdb file it prodcues. Cheers, Boaz - Original Message - From: Tim Gruene Date: Tuesday, November 2, 2010 16:59 Subject: Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues To: CCP4BB@JISCMAIL.AC.UK > Hello Buz, > > I do not know what you mean by 'linear map', but according to > its manual, the > ccp4-program "surface" writes a list of accessible are per atom > per residue, > which you could convert into the total fraction per residue with > not too much > effort. > > Is this what you are looking for? > > Cheers, Tim > > On Tue, Nov 02, 2010 at 10:36:56AM -0400, Buz Barstow wrote: > > Dear All, > > > > I'm looking for a software program to produce, given a 3D > atomic structure of a molecule, a linear map showing the surface > accessibility of residues in a protein structure. > > > > Would any one know of a program that can produce this sort of map. > > > > Thanks! and all the best, > > > > --Buz > > -- > -- > Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > phone: +49 (0)551 39 22149 > > GPG Key ID = A46BEE1A > > 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
[ccp4bb] Glutathione sepharose
Hello, For various reasons we are frequently expressing proteins with a GST tag. The glutathione sepharose beads that we are using for affinity purification seem to be difficult to regenerate and we see much lower capacity when used the second time. We are following the manufacturer's instructions for regeneration but this not very effective process as opposed to NiNTA which can regenerate multiple times. Due to this, the purification of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that can be successfully regenerated several times? Any comments will be greatly appreciated. Mirek
[ccp4bb] on the same note
On the same note with Mirek: does anyone know of a source other than GE for resin for purification of FLAG-ed proteins? Thanks. Vaheh -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mirek Cygler Sent: Tuesday, November 02, 2010 11:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Glutathione sepharose Hello, For various reasons we are frequently expressing proteins with a GST tag. The glutathione sepharose beads that we are using for affinity purification seem to be difficult to regenerate and we see much lower capacity when used the second time. We are following the manufacturer's instructions for regeneration but this not very effective process as opposed to NiNTA which can regenerate multiple times. Due to this, the purification of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that can be successfully regenerated several times? Any comments will be greatly appreciated. Mirek To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] on the same note
A2220 Sigma ANTI-FLAG® M2 Affinity Gel On Nov 2, 2010, at 11:01 AM, Oganesyan, Vaheh wrote: > On the same note with Mirek: does anyone know of a source other than GE for > resin for purification of FLAG-ed proteins? > > Thanks. > > Vaheh > > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mirek > Cygler > Sent: Tuesday, November 02, 2010 11:47 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Glutathione sepharose > > Hello, > For various reasons we are frequently expressing proteins with a GST > tag. The glutathione sepharose beads that we are using for affinity > purification seem to be difficult to regenerate and we see much lower > capacity when used the second time. We are following the manufacturer's > instructions for regeneration but this not very effective process as opposed > to NiNTA which can regenerate multiple times. Due to this, the purification > of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that > can be successfully regenerated several times? Any comments will be greatly > appreciated. > > Mirek > > > > To the extent this electronic communication or any of its attachments contain > information that is not in the public domain, such information is considered > by MedImmune to be confidential and proprietary. This communication is > expected to be read and/or used only by the individual(s) for whom it is > intended. If you have received this electronic communication in error, > please reply to the sender advising of the error in transmission and delete > the original message and any accompanying documents from your system > immediately, without copying, reviewing or otherwise using them for any > purpose. Thank you for your cooperation.
Re: [ccp4bb] Crystal gel band
I recently ran mass spec analysis on some crystals that I had obtained from an optimization screen. I was looking for modifications in the protein. In order to get enough signal, I had to harvest and dissolve about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer in order to get a strong enough signal for the experiment. Alas, I did not find the modification I had been looking for, but I am not surprised that one single crystal, or even a small cluster of them did not show a band on a coomassie-stained gel. Do you A) have a MS handy, and B) can you sacrifice ~8 crystals to get enough S/N for a good experiment? Good luck! From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Tuesday, November 02, 2010 10:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal gel band It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Crystal gel band
Hi Ivan, there are several tests (e.g. Izit dye, crush test) you can do discern protein from salt crystals but what was always very informative to me (and certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the crystals using the following protocol: - select a drop which contains some substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother liquor containing a 10% higher concentration of precipitant) - transfer all the crystalline material from the drop into the PCR-tube using a pipet (use stabilizing buffer from the PCR tube to collect all crystals) - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope. They should be at the bottom of the PCR-tube. - Remove as much as supernatant as you can (make sure not to remove your crystals), add stabilizing buffer to wash the crystals, and centrifuge again - repeat this washing protocol a few times - after the final washing step, add Laemli-buffer to the crystals and use this sample to load the SDS-PAGE gel - include a positive (eg. solubilize another drop directly in Laemli-buffer) and a negative (final washing buffer) control - use silver staining to visualize the protein This always work for me. If you don't see a band a this point I would be worried that it is salt. You could then choose to do a Western blot in stead of silver staining to increase the sensitivity. Make your to include control samples then. Kind regards, Kenneth Verstraete L-PROBE Ghent University Belgium Citeren "xaravich ivan" : Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
Re: [ccp4bb] Glutathione sepharose
Hi, Well you did not mention which company makes your GST sepharose, but I used GE GST Sepharose 4B quite often and was able to reuse it quite a few times. Chris On Tue, 2010-11-02 at 11:46 -0400, Mirek Cygler wrote: > Hello, > For various reasons we are frequently expressing proteins with a GST > tag. The glutathione sepharose beads that we are using for affinity > purification seem to be difficult to regenerate and we see much lower > capacity when used the second time. We are following the manufacturer's > instructions for regeneration but this not very effective process as opposed > to NiNTA which can regenerate multiple times. Due to this, the purification > of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that > can be successfully regenerated several times? Any comments will be greatly > appreciated. > > Mirek -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues
Hi, You first need a program to calculate ASA, e.g. Naccess. Once you have the ASA on a per-residue basis, you can plot a histogram, by e.g. feeding the data to a spreadsheet application of your choice. What is usually done is to list residues sequencewise vs. ASA. Best success, Nadir -- Pr. Nadir T. Mrabet Structural & Molecular Biochemistry INSERM U-954 UHP - Nancy 1, School of Medicine 54505 Vandoeuvre-les-Nancy Cedex France Tel : +33 (0)3.83.68.32.73 Fax : +33 (0)3.83.68.32.79 E-mail : nadir.mra...@medecine.uhp-nancy.fr Selon Buz Barstow : > Dear All, > > I'm looking for a software program to produce, given a 3D atomic structure of > a molecule, a linear map showing the surface accessibility of residues in a > protein structure. > > Would any one know of a program that can produce this sort of map. > > Thanks! and all the best, > > --Buz >
Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues
Try RasMol 2.7.5, e.g. load ../data/pdb1w0k.ent restrict not hoh map generate LRsurf dots map select atom within 1.8 show selected = Herbert J. Bernstein, Professor of Computer Science Dowling College, Kramer Science Center, KSC 121 Idle Hour Blvd, Oakdale, NY, 11769 +1-631-244-3035 y...@dowling.edu = On Tue, 2 Nov 2010, Buz Barstow wrote: Dear All, I'm looking for a software program to produce, given a 3D atomic structure of a molecule, a linear map showing the surface accessibility of residues in a protein structure. Would any one know of a program that can produce this sort of map. Thanks! and all the best, --Buz
[ccp4bb] UV microscopes for Xtal visualization
I have read the paper by Gill in Acta Crys F66: 364-372 and wonder what other peoples experience has been with the MUVIS from Formulatrix and the JANsci microscope for imaging 96 well plates. The JANsci instrument seems to have an advantage over the MUVIS in having two objectives. For detecting small xtals in LCP will the low field objective of the MUVIS be useful? Thanks, Mark --
Re: [ccp4bb] on the same note
Hi, There is no way you can compare NiNTA and GSH affinity supports. It is a lot easier to regenerate NiNTA. This has to do with the ligand, not the support. Kd is 10-100 times lower for GSH. GSH contains a "highly" reactive SH group if enough thiolate is present (oxydation and/or nucleophilic attack). It is recommanded to avoid hi pH and the presence of metal ions. HTH, Nadir -- Pr. Nadir T. Mrabet Structural & Molecular Biochemistry INSERM U-954 UHP - Nancy 1, School of Medicine 54505 Vandoeuvre-les-Nancy Cedex France Tel : +33 (0)3.83.68.32.73 Fax : +33 (0)3.83.68.32.79 E-mail : nadir.mra...@medecine.uhp-nancy.fr Selon "Oganesyan, Vaheh" : > On the same note with Mirek: does anyone know of a source other than GE for > resin for purification of FLAG-ed proteins? > > Thanks. > > Vaheh > > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mirek > Cygler > Sent: Tuesday, November 02, 2010 11:47 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Glutathione sepharose > > Hello, > For various reasons we are frequently expressing proteins with a GST > tag. The glutathione sepharose beads that we are using for affinity > purification seem to be difficult to regenerate and we see much lower > capacity when used the second time. We are following the manufacturer's > instructions for regeneration but this not very effective process as opposed > to NiNTA which can regenerate multiple times. Due to this, the purification > of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that > can be successfully regenerated several times? Any comments will be greatly > appreciated. > > Mirek > > > > To the extent this electronic communication or any of its attachments contain > information that is not in the public domain, such information is considered > by MedImmune to be confidential and proprietary. This communication is > expected to be read and/or used only by the individual(s) for whom it is > intended. If you have received this electronic communication in error, > please reply to the sender advising of the error in transmission and delete > the original message and any accompanying documents from your system > immediately, without copying, reviewing or otherwise using them for any > purpose. Thank you for your cooperation. >
Re: [ccp4bb] What makes the difference between 2 composite omit maps?
Thanks! Can you refer me some documents about your following statements:
derivation of sigmaa-weighted 2mFo-DFc formula is by calculating Fourier
coefficients of the following map:
Rescaled composite omit map, where minimal structural element (of the size
about the resolution element) is being omitted and the starting point is
the map with coefficients m*Fo*exp(i*phiCalc)
It seems the above was not involved in Read's publications about SIGMAA.
Thanks again!
Hailiang
> sigmaa-weighted 2mFo-DFc is the _COMPOSIT_OMIT_ map. There is no point in
> calculating omit map of an omit map
>
> A brief explanation: derivation of sigmaa-weighted 2mFo-DFc formula is by
> calculating Fourier coefficients of the following map:
>
> Rescaled composite omit map, where minimal structural element (of the size
> about the resolution element) is being omitted and the starting point is
> the map with coefficients m*Fo*exp(i*phiCalc)
>
> BTW, composite omit map of a map with coefficients Fo*exp(i*phiCalc) is
> simply Fo-1/2Fc map that after factor of 2 scaling becomes 2Fo-Fc map
>
>> Hi,
>> I want to calculate the sigmaa-weighted 2mFo-DFc composite omit map, and
> tried the following 2 scripts:
>>
>> (1)
>> ./omit hklin ${f}.mtz mapout ${f}.map <> LABI FP=mFo FC=DFC PHI=PHIC
>> RESO 29.50 3.22
>> SCAL 2.0 -1.0
>> EOF
>>
>> (2)
>> ./omit hklin ${f}.mtz mapout ${f}.map <> LABI FP=FWT FC=FC PHI=PHIC
>> RESO 29.50 3.22
>> SCAL 1.0 0.0
>> EOF
>>
>> The output maps are just different, and I wonder why. I am also more
> concerned about which one is more appropriate for the sigmaa-weighted
> 2mFo-DFc composite omit map.
>>
>> (mFo is what I generated from the SIGMAA output)
>>
>> Thanks for any suggestions!
>>
>> Best Regards, Hailiang
>>
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385
> Fax. 214-645-6353
>
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385
> Fax. 214-645-6353
>
>
Re: [ccp4bb] Glutathione sepharose
I am using the glutathione sepharose 4B beads from GE. I had noticed that the capture efficiency does decrease with usage, though I have successfully regenerated the beads at least 20 times and I am still using them. I regenerate using 2CV of 6M guanidine after each run and every 5 runs with 70% ethanol as well. I have also used 5CV of 1M NaCl and noticed elution of proteins. I perform these experiments using the gravity flow method with a glass column. I have noted that the beads do clump together. A gentle re-suspension using a pipette seems to be good at unclumping them and improve capture. Also the diameter of the column seems to be important. I have noticed that using 5ml of beads on a 1cm diameter column captures far less than 5ml of beads on a 2.5cm diameter column. Hope this helps!
Re: [ccp4bb] Crystal gel band
Thanks all of you for your answers. As you guessed I was doing coomassie staining from single crystal (0.10-0.13-0.05).Fortunately I had lots of not so great looking single crystals from similar drops and I took about 10-12 of them.Then I could see a faint band where I was expecting! Thanks again. Ivan On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete < kenneth.verstra...@ugent.be> wrote: > Hi Ivan, > > there are several tests (e.g. Izit dye, crush test) you can do discern > protein from salt crystals but what was always very informative to me (and > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the > crystals using the following protocol: > > - select a drop which contains some substantial crystalline material. The > crystals can be many and small (crystal shower) or few and large. > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother > liquor containing a 10% higher concentration of precipitant) > - transfer all the crystalline material from the drop into the PCR-tube > using a pipet (use stabilizing buffer from the PCR tube to collect all > crystals) > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the > crystals under the microscope. They should be at the bottom of the PCR-tube. > - Remove as much as supernatant as you can (make sure not to remove your > crystals), add stabilizing buffer to wash the crystals, and centrifuge again > - repeat this washing protocol a few times > - after the final washing step, add Laemli-buffer to the crystals and use > this sample to load the SDS-PAGE gel > - include a positive (eg. solubilize another drop directly in > Laemli-buffer) and a negative (final washing buffer) control > - use silver staining to visualize the protein > > This always work for me. If you don't see a band a this point I would be > worried that it is salt. You could then choose to do a Western blot in stead > of silver staining to increase the sensitivity. Make your to include control > samples then. > > Kind regards, > > Kenneth Verstraete > L-PROBE > Ghent University > Belgium > > > Citeren "xaravich ivan" : > > > Hi everyone, >> I have grown some crystals after micro-seeding starting from thin-small >> needles from needle-clusters. These crystals are larger in size than the >> needles but are comparable to the shape and don't look like salt crystals. >> But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a >> home source,handy and would like to send these to the synchrotron. >> >> Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, >> the amount of protein is < 1uG? >> Has anyone experienced such a thing (no band in gel, but crystal >> diffracts)? >> >> It would be nice if I get observations/suggestions. >> >> ivan >> >> > > >
[ccp4bb] Lysis of Pichia pastoris
Hello all; I have successfully expressed a membrane protein in Pichia pastoris, however I am having a difficult time with cell lysis. I have used a Avestin emulsiflex to lyse them, however I have had many difficulties with the system clogging up, and parts wearing out with the high pressures. So I am wondering what other people out there use to lyse their Pichia? In particular we have been considering a microfluidizer a Retsch mixer mill a TS-series cell disruptor (Constans systems) Any thoughts on these options, or other systems would be much appreciated! Best regards, Cory Cory Brooks, Ph.D. Postdoctoral Fellow University of Alberta
Re: [ccp4bb] Lysis of Pichia pastoris
You shouldn't have clogging up problems in the first place. We don't use Pichia but have the same system and I've used it before at the MPI in Martinsried. The system is designed that there are no parts which break, you only have to maintain it on a regular basis and clean it properly. Do you have a manual or pressure driven controller ? With the manual one, people tend to over tighten the valve and thereby ruining the system as a metal spike hits a metal outlet (in the end you grind a hole into the base and can break the whole system). That's really the only part that can be broken. With the pressure controlled system this user error is eliminated. Take it apart, clean it and reassemble it is my suggestion - did you ever took it apart to clean it up or add the vacuum lubricant ? By the way the Hampton heavy duty vacuum grease works very well for that purpose, needs to be replaced every three to four months. The only other alternative would be a glass beater and regularly autoclave and clean the glass beads. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Nov 2, 2010, at 5:33 PM, Cory Brooks wrote: > Hello all; > > I have successfully expressed a membrane protein in Pichia pastoris, > however I am having a difficult time with cell lysis. > I have used a Avestin emulsiflex to lyse them, however I have had many > difficulties with the system clogging up, and parts wearing out with the > high pressures. > > So I am wondering what other people out there use to lyse their Pichia? > In particular we have been considering > a microfluidizer > a Retsch mixer mill > a TS-series cell disruptor (Constans systems) > > Any thoughts on these options, or other systems would be much appreciated! > > Best regards, > Cory > > Cory Brooks, Ph.D. > Postdoctoral Fellow > University of Alberta
Re: [ccp4bb] Lysis of Pichia pastoris
I know this seems a bit crazy, but I used to lyse s. cerevisiae by pelleting the cells into a 50mL conical, freezing in liquid nitrogen, and using a power drill with a drill bit fitting the caliber of the tube, all at liquid nitrogen temp through periodic (or constant) re-immersion in nitrogen. It seemed to work really well, and it was nice how no proteolysis could take place because of the temp. It yielded a talc-like powder which, once all nitrogen was gone, was resuspended in whatever buffer. This can also be done for e coli pellets, and might be advisable for really proteolysis-sensitive proteins. JPK On Tue, Nov 2, 2010 at 4:33 PM, Cory Brooks wrote: > Hello all; > > I have successfully expressed a membrane protein in Pichia pastoris, > however I am having a difficult time with cell lysis. > I have used a Avestin emulsiflex to lyse them, however I have had many > difficulties with the system clogging up, and parts wearing out with the > high pressures. > > So I am wondering what other people out there use to lyse their Pichia? > In particular we have been considering > a microfluidizer > a Retsch mixer mill > a TS-series cell disruptor (Constans systems) > > Any thoughts on these options, or other systems would be much appreciated! > > Best regards, > Cory > > Cory Brooks, Ph.D. > Postdoctoral Fellow > University of Alberta >
Re: [ccp4bb] Lysis of Pichia pastoris
Yes that's how it's conventionally done if you are working with plants. Instead of a drill (what did safety inspection said to that, did you have proper precautions in place etc.) they use a mortar cooled in LN2 and also containing LN2 within the mortar and a suitable grinder plus cryo gloves (for you). Then you keep grinding until you have a nice pesto. Thinking about this, we should do that for smaller amounts instead of diluting the cells so that they can be run through the Emulsiflex ... Need to go to my freezer and pull out a pellet and grind them up. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Nov 2, 2010, at 6:19 PM, Jacob Keller wrote: > I know this seems a bit crazy, but I used to lyse s. cerevisiae by > pelleting the cells into a 50mL conical, freezing in liquid nitrogen, > and using a power drill with a drill bit fitting the caliber of the > tube, all at liquid nitrogen temp through periodic (or constant) > re-immersion in nitrogen. It seemed to work really well, and it was > nice how no proteolysis could take place because of the temp. It > yielded a talc-like powder which, once all nitrogen was gone, was > resuspended in whatever buffer. This can also be done for e coli > pellets, and might be advisable for really proteolysis-sensitive > proteins. > > JPK > > On Tue, Nov 2, 2010 at 4:33 PM, Cory Brooks wrote: >> Hello all; >> >> I have successfully expressed a membrane protein in Pichia pastoris, >> however I am having a difficult time with cell lysis. >> I have used a Avestin emulsiflex to lyse them, however I have had many >> difficulties with the system clogging up, and parts wearing out with the >> high pressures. >> >> So I am wondering what other people out there use to lyse their Pichia? >> In particular we have been considering >> a microfluidizer >> a Retsch mixer mill >> a TS-series cell disruptor (Constans systems) >> >> Any thoughts on these options, or other systems would be much appreciated! >> >> Best regards, >> Cory >> >> Cory Brooks, Ph.D. >> Postdoctoral Fellow >> University of Alberta >>
Re: [ccp4bb] Lysis of Pichia pastoris
Hi Cory, I am afraid to say that Pichia and Saccharomyces are tough cells to break compared to bacteria. We also purifiy membrane proteins in yeast in my lab so we have gone through this process. With regular yeast we can break in an emulsiflex but it takes some hard work and this is not really viable option on a large scale once you process several liters of cell culture. So we use a bead-beater ( from BIOSPEC) and glass beads (0.5 mm for yeast, the diameter depends on the kind of organism you want to disrupt). it works well and is quite fast , however you have to deal with the bead cleaning part and the losses due to the mass of liquid trapped in the beads (rinsing of beads with buffer is fine but it results in an increase burden at the membrane centrifugation step) It takes some optimization (mass of beads/mass of cells processed), but once this is is set this is probably the way to go, Bead beaters come in various sizes depending on the volumes you want to process. It is just a blender after all. Hope this helps. Best regards -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] What makes the difference between 2 composite omit maps?
I am not aware of this point being explicitly made. Maybe somebody else
could point to the relevant reference?
However, the logic here is very simple:
(1) Take a model and generate from it Fc
(2) Calculate map with minimal rms error m*Fo*exp(i*phiCalc)
(3) This map is biased with respect to errors in the model
(4) To avoid this bias one can subtract a part of the model
(Fc*coefficient) from the above map
- the coefficient is chosen so electron density in the resulting map
does not change at the point where we added or subtracted an atom;
- conceptually the above procedure when we subtract an atom is the
composite omit map
- "does not change" means here: within first order approximation, we
ignore second order effects
- for sigmaA-weighted map this coefficient is D/2
(5) The subtraction gives: (m*Fo-D/2*Fc)exp(i*phiCalc)
(6) After multiplication by 2, we get (2m*Fo-DFc)exp(i*phiCalc)
Hailiang Zhang wrote:
Thanks! Can you refer me some documents about your following statements:
derivation of sigmaa-weighted 2mFo-DFc formula is by calculating Fourier
coefficients of the following map:
Rescaled composite omit map, where minimal structural element (of the size
about the resolution element) is being omitted and the starting point is
the map with coefficients m*Fo*exp(i*phiCalc)
It seems the above was not involved in Read's publications about SIGMAA.
Thanks again!
Hailiang
sigmaa-weighted 2mFo-DFc is the _COMPOSIT_OMIT_ map. There is no point in
calculating omit map of an omit map
A brief explanation: derivation of sigmaa-weighted 2mFo-DFc formula is by
calculating Fourier coefficients of the following map:
Rescaled composite omit map, where minimal structural element (of the size
about the resolution element) is being omitted and the starting point is
the map with coefficients m*Fo*exp(i*phiCalc)
BTW, composite omit map of a map with coefficients Fo*exp(i*phiCalc) is
simply Fo-1/2Fc map that after factor of 2 scaling becomes 2Fo-Fc map
Hi,
I want to calculate the sigmaa-weighted 2mFo-DFc composite omit map, and
tried the following 2 scripts:
(1)
./omit hklin ${f}.mtz mapout ${f}.map <
concerned about which one is more appropriate for the sigmaa-weighted
2mFo-DFc composite omit map.
(mFo is what I generated from the SIGMAA output)
Thanks for any suggestions!
Best Regards, Hailiang
Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353
Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353
--
Zbyszek Otwinowski
UT Southwestern Medical Center
5323 Harry Hines Blvd., Dallas, TX 75390-8816
(214) 645 6385 (phone) (214) 645 6353 (fax)
zbys...@work.swmed.edu
Re: [ccp4bb] Lysis of Pichia pastoris
I tried the mortar procedure, and found it took forever, with little chips of thawing yeast inevitably hopping out and sticking wherever, since everything was so brittle, even though I had extruded the pre-frozen pellet through a syringe into liq N2 to make yeast-pellet-noodles first. In contrast, the drill procedure actually was not messy at all, since mostly the stuff was powdered and contained in the tube, so I would drill, empty, drill, empty, etc. I used cryogloves for holding the tube, and the spade-type drill bit was ~$5 at Home Depot. Jacob On Tue, Nov 2, 2010 at 5:29 PM, Jürgen Bosch wrote: > Yes that's how it's conventionally done if you are working with plants. > Instead of a drill (what did safety inspection said to that, did you have > proper precautions in place etc.) they use a mortar cooled in LN2 and also > containing LN2 within the mortar and a suitable grinder plus cryo gloves > (for you). > Then you keep grinding until you have a nice pesto. Thinking about this, we > should do that for smaller amounts instead of diluting the cells so that > they can be run through the Emulsiflex ... Need to go to my freezer and pull > out a pellet and grind them up. > Jürgen > - > Jürgen Bosch > Johns Hopkins Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Phone: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-3655 > http://web.mac.com/bosch_lab/ > On Nov 2, 2010, at 6:19 PM, Jacob Keller wrote: > > I know this seems a bit crazy, but I used to lyse s. cerevisiae by > pelleting the cells into a 50mL conical, freezing in liquid nitrogen, > and using a power drill with a drill bit fitting the caliber of the > tube, all at liquid nitrogen temp through periodic (or constant) > re-immersion in nitrogen. It seemed to work really well, and it was > nice how no proteolysis could take place because of the temp. It > yielded a talc-like powder which, once all nitrogen was gone, was > resuspended in whatever buffer. This can also be done for e coli > pellets, and might be advisable for really proteolysis-sensitive > proteins. > > JPK > > On Tue, Nov 2, 2010 at 4:33 PM, Cory Brooks wrote: > > Hello all; > > I have successfully expressed a membrane protein in Pichia pastoris, > > however I am having a difficult time with cell lysis. > > I have used a Avestin emulsiflex to lyse them, however I have had many > > difficulties with the system clogging up, and parts wearing out with the > > high pressures. > > So I am wondering what other people out there use to lyse their Pichia? > > In particular we have been considering > > a microfluidizer > > a Retsch mixer mill > > a TS-series cell disruptor (Constans systems) > > Any thoughts on these options, or other systems would be much appreciated! > > Best regards, > > Cory > > Cory Brooks, Ph.D. > > Postdoctoral Fellow > > University of Alberta > > >
