[ccp4bb] ccp4mg question
Dear all, I am working with a cyclic protein and I am trying to make a figure with ccp4mg. I would like to know how to say to ccp4mg that the N and C-terminus are bound Although atoms are at a covalent bond distance, the chain is broken by ccp4mg... Many thanks in advance for your suggestions and help! Maria
Re: [ccp4bb] ccp4mg question
possible ugly workaround: renumber the pdb file so where ccp4mg thinks the N and C-termini are is hidden in the image you are making (if possible) if you want to make different views you may need to make differently renumbered pdb files. (but probably other people have a smarter way) as an aside, PyMol I think does not have these problems, at least we make images of cyclic peptides with it and I haven't run into it. Quoting SANCHEZ BARRENA, MARIA JOSE: Dear all, I am working with a cyclic protein and I am trying to make a figure with ccp4mg. I would like to know how to say to ccp4mg that the N and C-terminus are bound Although atoms are at a covalent bond distance, the chain is broken by ccp4mg... Many thanks in advance for your suggestions and help! Maria Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] ccp4mg question
On 15/11/2012 12:26, SANCHEZ BARRENA, MARIA JOSE wrote: Dear all, I am working with a cyclic protein and I am trying to make a figure with ccp4mg. I would like to know how to say to ccp4mg that the N and C-terminus are bound Although atoms are at a covalent bond distance, the chain is broken by ccp4mg... Many thanks in advance for your suggestions and help! Maria Dear Maria, On e thing to try is to click on the icon next to the molecule name (in the display table). Select Structute definition - Edit bonds. A new window should appear. In the main graphics window right click on one of the atoms you wish to connect and select Add/delete bond - first atom. Then right click on secon atom, select Add/delete bond - (name of first atom). This should draw a bond between them. Best wishes, Stuart McNicholas
Re: [ccp4bb] ccp4mg question
Dear all, it seems that none of the tricks from Mark or Tim or Stuart´s ccp4mg making bond strategy works out... In all cases, the bond is made when drawing in think/fat band style... However, when representing in ribbon style, the link is not there... Any other suggestion is welcome! Cheers, Maria Quoting Stuart McNicholas: On 15/11/2012 12:26, SANCHEZ BARRENA, MARIA JOSE wrote: Dear all, I am working with a cyclic protein and I am trying to make a figure with ccp4mg. I would like to know how to say to ccp4mg that the N and C-terminus are bound Although atoms are at a covalent bond distance, the chain is broken by ccp4mg... Many thanks in advance for your suggestions and help! Maria Dear Maria, On e thing to try is to click on the icon next to the molecule name (in the display table). Select Structute definition - Edit bonds. A new window should appear. In the main graphics window right click on one of the atoms you wish to connect and select Add/delete bond - first atom. Then right click on secon atom, select Add/delete bond - (name of first atom). This should draw a bond between them. Best wishes, Stuart McNicholas
Re: [ccp4bb] ccp4mg question
On 15/11/2012 13:57, SANCHEZ BARRENA, MARIA JOSE wrote: Dear all, it seems that none of the tricks from Mark or Tim or Stuart´s ccp4mg making bond strategy works out... In all cases, the bond is made when drawing in think/fat band style... However, when representing in ribbon style, the link is not there... Any other suggestion is welcome! Cheers, Maria Dear Maria, Ribbons will fail as you say. This is a case I had never before considered. A chain is considered to have a start and end and I will have to think a little how to address this issue. It will take a day or so to fix this in CCP4MG. Mark's suggestion would seem to be the only solution at the moment. Make two copies of your PDB file and move the end residue to the beginning in one of the copies and renumber the residiue to be first in sequence. The show both structures, you may get something acceptible. Best wishes, Stuart
[ccp4bb] Postdoctoral position
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Re: [ccp4bb] vitrification vs freezing
Dear Sebastiano, This is not entirely straight-forward. The Oxford English dictionary gives the first definition of freeze relevant to this discussion as: Of (a body of) water: be converted into or become covered with ice through loss of heat This is certainly not what we want to do to our crystals. However, another definition in OED is: Cause (a liquid) to solidify by removal of heat, suggesting that this does not necessarily mean the formation of crystals. The Larousse Dictionary of Science and Technology (1995) has the following definition: Freeze-drying (Biol.) A method of fixing tissues sufficiently rapidly as to inhibit the formation of ice-crystals. The Dictionary of Microbiology and Molecular Biology (3rd Ed) in the entry on Freezing has the sentence: Rapid freezing tends to prevent the ice crystal formation by encouraging vitrification. Both of these erstwhile volumes therefore suggest that freezing does not necessarily imply the formation of crystals. However, the term is ambiguous, while vitrification is not. Personally I use cryocooled instead. Best wishes, Andrew On 15 Nov 2012, at 17:13, Sebastiano Pasqualato wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu
Re: [ccp4bb] vitrification vs freezing
Hi Sebastiano, Elspeth Garman howls bloody murder everytime someone says they froze their crystals. I think her issue is with the description of the process of successfully flashcooling crystals in the presence of cryoprotectants as freezing. Freezing technically is understood to imply the formation of hexagonal ice while what one really means is the successful solidification of water in a random orientation (vitrification) and the prevention of the hexagonal ice. Semantics semantics! I'd stick with flashcooled or something along those lines. Raji On Thu, Nov 15, 2012 at 12:13 PM, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] vitrification vs freezing
On Thursday, November 15, 2012 09:13:58 am you wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? What we aim for is vitrification: to make into a glass. What we achieve is another matter. Sometimes dipping into LN2 produces a partially ordered (non-glasslike) state in the solvent that is bad for our diffraction experiment. Either result, the desired glass or the unfortunately crystalline ice, is an example of freezing: to make into a solid by removing heat. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] vitrification vs freezing
s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N. On 11/15/2012 11:59 AM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear s, I have heard this discussion before and reminds me of people claiming strawberries were nuts - which botanically may be correct, but would still not make me complain about strawberries in a fruit cake I ordered at a restaurant. My Pengiun English Dictionary states (amongst other explanations) freeze: to make extremely cold, so as long as you think your article is written in English, you did not say anything wrong, assuming your readers are intelligent enough to understand what you are trying to say - and in a crystallographic article, the process of 'freezing' your crystal is most likely not your main point where you need to be 100% unambiguous. Cheers, Tim On 11/15/2012 06:13 PM, Sebastiano Pasqualato wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQpS17UxlJ7aRr7hoRAvX0AJ9b3YYQ4kXu5J0wJdEYudPclTmKtQCg8HSx R4wgkmbp2l7Q/ns/HfJkqgY= =R9Wp -END PGP SIGNATURE- -- Ruslan Sanishvili (Nukri) Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov
Re: [ccp4bb] vitrification vs freezing
Dear Andrew, I would suggest that Larousse may need to revisit their entry - freeze-drying (in every context I have come across it) refers to lyophilisation, which (i) specifically requires the formation of ice crystals, and (ii) results in the removal of all of the resulting ice from the sample. Tom This is certainly not what we want to do to our crystals. However, another definition in OED is: Cause (a liquid) to solidify by removal of heat, suggesting that this does not necessarily mean the formation of crystals. The Larousse Dictionary of Science and Technology (1995) has the following definition: Freeze-drying (Biol.) A method of fixing tissues sufficiently rapidly as to inhibit the formation of ice-crystals. The Dictionary of Microbiology and Molecular Biology (3rd Ed) in the entry on Freezing has the sentence: Rapid freezing tends to prevent the ice crystal formation by encouraging vitrification. Both of these erstwhile volumes therefore suggest that freezing does not necessarily imply the formation of crystals. However, the term is ambiguous, while vitrification is not. Personally I use cryocooled instead. Best wishes, Andrew On 15 Nov 2012, at 17:13, Sebastiano Pasqualato wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
Re: [ccp4bb] vitrification vs freezing
Perhaps it's an artisan organic locavore fruit cake. Either way, your *crystal* is not vitrified. The solvent in your crystal might be glassy but your protein better still hold crystalline order (cf. ice) or you've wasted your time. Ergo, cryo-cooled is the description to use. Phil Jeffrey Princeton On 11/15/12 1:14 PM, Nukri Sanishvili wrote: s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N.
Re: [ccp4bb] relations between groups and subgroups?
Everyone knows that there are 230 space groups, and these belong to one
of 32 point groups, which, in turn, belong to one of the 14 Bravais
lattices, and 7 crystal systems: triclinic, monoclinic, orthorhombic,
tetragonal, hexagonal, rhombohedral and cubic.
Or are there? If you look in ${CLIBD}/symop.lib of your nearest CCP4
Suite install, you will find not 230 but 266 entries for space groups,
and 43 different kinds of point groups. And those so-called
rhombohedral systems can apparently be represented as hexagonal, so
maybe there are only six crystal systems?
Blasphemy! (I can almost hear the purists now) But, the point I am
trying to make here is that there is a disconnect between the
traditional way that crystallography is taught (aka Chapter 1: crystal
symmetry) and the pragmatic practice of crystallography (aka what
MOSFLM is doing). It is ironic really that the first thing you must
decide for a new crystal is its space group when in reality it is the
last thing you will ever be certain about it. Probably one of the most
common examples of this is the P2221 and P21212 space groups.
Technically, P2122, P2212, etc are NOT space groups! However, given
that orthorhombic unit cells are traditionally sorted abc, simply
giving such a unit cell with the space group P2221 is not enough
information to be sure which axis is the screwy one. Also, I'm sure
many of you have noticed that for any trigonal/hexagonal crystal there
is always a C222 cell that comes up in autoindexing? This is because
you can always index a trigonal lattice along a diagonal and that
makes it look like centered orthorhombic. But, if you try going with
that C222 choice you find that it doesn't merge ... most of the time.
The fact of the matter is that all autoindexing algorithms give you is a
unit cell, and that is just six numbers. The cell dimensions generally
allow you to EXCLUDE a great many symmetry operations, but they can
never really INFER symmetry. Except, of course, in the special case
where all three angles of the reduced cell are not 90 (or 60) degrees,
then the only possible space group is P1. On the other hand, it is
perfectly possible to have P1 symmetry with all three cell edges the
same length and all angles 90 degrees. It just isn't very likely (in
the posterior probability sense). This is why MOSFLM and other
autoindexing programs pick the highest-symmetry lattice and give you a
space group consistent with that lattice, even though there are plenty
of other possibilities. This is why you should always take the space
group that comes out of autoindexing with a grain of salt. Do NOT make
the mistake of classifying your crystals by the result of autoindexing
alone!
Something similar is true for point groups. A high Rsym for a given
symmetry operator (like you will see in the output of pointless) means
that there is NO WAY that the given symmetry operation is part of the
space group. A low Rsym, however, does not mean that you have a given
symmetry. Could always be some kind of twinning or
nearly-crystallographic NCS (NCNCS?). Twinning is relatively rare, and
gets increasingly rare as you get into the non-merohedral stuff, but it
is always a possibility. Yes, intensity statistics can tell you
something is twinned, but if you have just the right mixture of twinning
and pseudotranslation, then the twinning can go undetected. So, in
general, you can always have _less_ symmetry than you think, but proving
the existence of a symmetry operation is hard.
Space groups, or narrowing down the screw vs rotation nature of various
axes generally requires phasing and looking at a map. The one with
right-handed alpha helices is the correct one. Yes, there are plenty of
tricks like systematic absences, native Pattersons and the like but
there are a lot of false positives and false negatives possible with
each of these. In fact, you tend to throw out more rejects in scaling
than you ever have observations of systematic absences, so why trust
those absent spots so much? In fact, sometimes you need to even go
all the way to the end of refinement to settle the space group. It is
possible to get stuck with R/Rfree too high because the crystal very
slightly violates the symmetry you think it has. (NCNCS again)
Whatever you do, don't forget to try all the possible P2122-like space
groups if you are searching for heavy atoms or doing MR with a primitive
orthorhombic crystal. Far too many people have missed solving their
structure because they didn't know to do this! Fortunately, modern
computers tend to have 8 or so CPUs in them, and there are never more
than 8 space groups possible on any given point group. So, you might as
well launch 8 parallel MR or heavy-atom site-finding jobs in different
space groups, since it will take just as long to run 8 jobs as it will
take to do only one. Well, okay, some of the non-protein ones have more
than 8
Re: [ccp4bb] vitrification vs freezing
Hi Tim, in the UK, you'd probably be rather surprised how many nuts your fruitcake contains, none of them strawberries (thus the saying as nutty as a fruitcake). Andreas On 15/11/2012 5:59, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear s, I have heard this discussion before and reminds me of people claiming strawberries were nuts - which botanically may be correct, but would still not make me complain about strawberries in a fruit cake I ordered at a restaurant. My Pengiun English Dictionary states (amongst other explanations) freeze: to make extremely cold, so as long as you think your article is written in English, you did not say anything wrong, assuming your readers are intelligent enough to understand what you are trying to say - and in a crystallographic article, the process of 'freezing' your crystal is most likely not your main point where you need to be 100% unambiguous. Cheers, Tim -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] vitrification vs freezing
On Thursday, November 15, 2012 10:14:54 am Raji Edayathumangalam wrote: Hi Sebastiano, Elspeth Garman howls bloody murder everytime someone says they froze their crystals. I think her issue is with the description of the process of successfully flashcooling crystals in the presence of cryoprotectants as freezing. Freezing technically is understood to imply the formation of hexagonal ice Not according to common English usage or any of the dictionaries I looked in. E.g. American Heritage Dictionary: Freeze 1.a. To pass from the liquid to the solid state by loss of heat. It needn't refer to water at all, although that is the most common context. You can find instructions for freezing olive oil to preserve it; when I lived in Madison one occassionally had to worry about frozen engine oil; a headline from earlier this year claimed Russian rivers clogged with frozen oil. while what one really means is the successful solidification of water in a random orientation (vitrification) and the prevention of the hexagonal ice. Semantics semantics! I'd stick with flashcooled or something along those lines. Raji Funny you should say that :-) While I have never had a referred complain about frozen crystals, I have had several complain that flash cooling is different from immersing in liquid nitrogen. I never figured out what they had in mind, but have since tried to avoid the term flash cooling. By the way, cryo-cooled must be a term advocated by The Department of Redundancy Department. cryo - From Greek kruos, icy cold Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] vitrification vs freezing
talking semantics, kruos (Κρυος), means just cold, not icy cold. Cold in Greece is not nearly icy. Unlike the Netherlands ... it only gets cold when its really icy ;-) Tassos On 15 Nov 2012, at 19:45, Ethan Merritt wrote: From Greek kruos, icy cold
Re: [ccp4bb] vitrification vs freezing
Hi Ethan, I am not a linguist of Greek or even of English but I would assume that the term cryo-cooling is advocated not by DRD but by the people who want to distinguish between cooling down to *cryogenic* temperatures and say, cooling from 25 C to 4 C. Cheers, N. On 11/15/2012 12:45 PM, Ethan Merritt wrote: On Thursday, November 15, 2012 10:14:54 am Raji Edayathumangalam wrote: Hi Sebastiano, Elspeth Garman howls bloody murder everytime someone says they froze their crystals. I think her issue is with the description of the process of successfully flashcooling crystals in the presence of cryoprotectants as freezing. Freezing technically is understood to imply the formation of hexagonal ice Not according to common English usage or any of the dictionaries I looked in. E.g. American Heritage Dictionary: Freeze 1.a. To pass from the liquid to the solid state by loss of heat. It needn't refer to water at all, although that is the most common context. You can find instructions for freezing olive oil to preserve it; when I lived in Madison one occassionally had to worry about frozen engine oil; a headline from earlier this year claimed Russian rivers clogged with frozen oil. while what one really means is the successful solidification of water in a random orientation (vitrification) and the prevention of the hexagonal ice. Semantics semantics! I'd stick with flashcooled or something along those lines. Raji Funny you should say that :-) While I have never had a referred complain about frozen crystals, I have had several complain that flash cooling is different from immersing in liquid nitrogen. I never figured out what they had in mind, but have since tried to avoid the term flash cooling. By the way, cryo-cooled must be a term advocated by The Department of Redundancy Department. cryo - From Greek kruos, icy cold Ethan -- Ruslan Sanishvili (Nukri) Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov
Re: [ccp4bb] vitrification vs freezing
Actually, there is a particular kind of freezing than can be a good thing: cubic ice. The specific volume of cubic ice is about 2% higher than that of amorphous solid water (or hyperquenched glassy water). In cases where the preferred specific volume of the protein lattice is a little bigger than the preferred specific volume of the stuff in the solvent channels upon vitrification, a small amount of cubic ice character can actually make your crystal diffract better. Basically, a mis-match in the preferred volume of the protein and solvent creates stress, and stress makes high-angle spots go away (see Juers et al. 2001, 2004). I think this is one of the most under-appreciated things about screening for cryos, and seems to be the main cause of that mythical one crystal in a thousand that diffracted well problem. In my experience, this means you are being very consistent in your cryo-cooling but you made one mistake. Final density upon flash-cooling is VERY sensitive to the cooling rate, and little things like the thickness of the layer of cold N2 gas above your liquid N2 can change the cooling rate by a factor of 100 or so (Warkentin et al. 2006). You can tell if you've got some cubic ice character by looking at the background of your diffraction pattern. Hexagonal ice has three rings at 3.45, 3.68, and 3.91 A, whereas cubic ice only has the 3.68 A ring. Moreover, noone has ever grown a macroscopic crystal of cubic ice, and the smaller the crystallites get, the broader the 3.68 A ring becomes. Eventually, it can be hard to tell the difference between nano-crystalline cubic ice and the diffuse background expected for amorphous solvent. You have to look carefully at the width of the water ring, because the first major diffuse ring from amorphous solid water is also at 3.68 A. In fact, all the major diffraction peaks of cubic ice are precisely centered on the diffuse rings of amorphous solid water. This is perhaps not a coincidence, but it does mean that there is a continuum of states between a sharp ice ring and an amorphous water ring. Your best diffraction may well be somewhere in the middle. -James Holton MAD Scientist On 11/15/2012 10:12 AM, A Leslie wrote: Dear Sebastiano, This is not entirely straight-forward. The Oxford English dictionary gives the first definition of freeze relevant to this discussion as: Of (a body of) water: be converted into or become covered with ice through loss of heat This is certainly not what we want to do to our crystals. However, another definition in OED is: Cause (a liquid) to solidify by removal of heat, suggesting that this does not necessarily mean the formation of crystals. The Larousse Dictionary of Science and Technology (1995) has the following definition: Freeze-drying (Biol.) A method of fixing tissues sufficiently rapidly as to inhibit the formation of ice-crystals. The Dictionary of Microbiology and Molecular Biology (3rd Ed) in the entry on Freezing has the sentence: Rapid freezing tends to prevent the ice crystal formation by encouraging vitrification. Both of these erstwhile volumes therefore suggest that freezing does not necessarily imply the formation of crystals. However, the term is ambiguous, while vitrification is not. Personally I use cryocooled instead. Best wishes, Andrew On 15 Nov 2012, at 17:13, Sebastiano Pasqualato wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu mailto:sebastiano.pasqual...@ieo.eu
Re: [ccp4bb] vitrification vs freezing
If Hakon Hope is reading this I can see his eyes rolling back in his head. I vote for cryo-cooling, since he was one of the inventors of this method, see the following abstract from his 1988 paper in Acta Cryst. B: Methods have been developed that allow facile X-ray data collection for biological macromolecules at cryogenic (near liquid N2) temperatures. The crystals are first transferred from their mother liquor to a hydrocarbon environment, then mounted with a standard glass fiber (no capillary), and flash cooled in situ with a cold nitrogen stream on the diffraction apparatus. This approach prevents freezing of the solvent in the crystals, so that they maintain their crystallographic integrity. Significant improvement of resolution can result from the cryogenic data collection, and radiation damage in the cooled crystals is greatly reduced, or eliminated, for the duration of data collection. Laurie Betts On Thu, Nov 15, 2012 at 1:58 PM, Nukri Sanishvili rsanishv...@anl.govwrote: Hi Ethan, I am not a linguist of Greek or even of English but I would assume that the term cryo-cooling is advocated not by DRD but by the people who want to distinguish between cooling down to *cryogenic* temperatures and say, cooling from 25 C to 4 C. Cheers, N. On 11/15/2012 12:45 PM, Ethan Merritt wrote: On Thursday, November 15, 2012 10:14:54 am Raji Edayathumangalam wrote: Hi Sebastiano, Elspeth Garman howls bloody murder everytime someone says they froze their crystals. I think her issue is with the description of the process of successfully flashcooling crystals in the presence of cryoprotectants as freezing. Freezing technically is understood to imply the formation of hexagonal ice Not according to common English usage or any of the dictionaries I looked in. E.g. American Heritage Dictionary: Freeze 1.a. To pass from the liquid to the solid state by loss of heat. It needn't refer to water at all, although that is the most common context. You can find instructions for freezing olive oil to preserve it; when I lived in Madison one occassionally had to worry about frozen engine oil; a headline from earlier this year claimed Russian rivers clogged with frozen oil. while what one really means is the successful solidification of water in a random orientation (vitrification) and the prevention of the hexagonal ice. Semantics semantics! I'd stick with flashcooled or something along those lines. Raji Funny you should say that :-) While I have never had a referred complain about frozen crystals, I have had several complain that flash cooling is different from immersing in liquid nitrogen. I never figured out what they had in mind, but have since tried to avoid the term flash cooling. By the way, cryo-cooled must be a term advocated by The Department of Redundancy Department. cryo - From Greek kruos, icy cold Ethan -- Ruslan Sanishvili (Nukri) Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667rsanishv...@anl.gov
Re: [ccp4bb] vitrification vs freezing
Isn't cryo-cooled redundant? James On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote: Perhaps it's an artisan organic locavore fruit cake. Either way, your *crystal* is not vitrified. The solvent in your crystal might be glassy but your protein better still hold crystalline order (cf. ice) or you've wasted your time. Ergo, cryo-cooled is the description to use. Phil Jeffrey Princeton On 11/15/12 1:14 PM, Nukri Sanishvili wrote: s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N.
Re: [ccp4bb] vitrification vs freezing
cryopreserved It says that the crystals were transferred to cryogenic temperatures in an attempt to increase their lifetime in the beam, and avoids all of the other problems with all of the other language described. I was really trying to stay out of this, because I understand what everyone means with all of their other word choices. On Nov 15, 2012, at 2:07 PM, James Stroud wrote: Isn't cryo-cooled redundant? James On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote: Perhaps it's an artisan organic locavore fruit cake. Either way, your *crystal* is not vitrified. The solvent in your crystal might be glassy but your protein better still hold crystalline order (cf. ice) or you've wasted your time. Ergo, cryo-cooled is the description to use. Phil Jeffrey Princeton On 11/15/12 1:14 PM, Nukri Sanishvili wrote: s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N.
Re: [ccp4bb] vitrification vs freezing
On 15/11/2012 20:15, James Stroud wrote: On Nov 15, 2012, at 10:59 AM, Tim Gruene wrote: I have heard this discussion before and reminds me of people claiming strawberries were nuts - which botanically may be correct, but would still not make me complain about strawberries in a fruit cake I ordered at a restaurant. My Pengiun English Dictionary states (amongst other explanations) freeze: to make extremely cold, Tim's comment strikes at the heart of the problem. I think the scientific community should decide a few points. 1. What is the approved language and dialect for science? 2. Within this dialect, what should be the authoritative dictionary? 3. Will we allow use of definitions that are not the primary definition (second, third, fourth). 4. Will we allow the use of homonyms? 5. If not, which homonyms should prevail? These are all very important questions if we completely disregard context in writing. James Hear hear! I never have the faintest idea what somebody means when they say I froze my crystals. They may as well be speaking Greek.
Re: [ccp4bb] vitrification vs freezing
Hi Sebastiano, I think the term vitrified crystal could be understood as a very nice oxymoron (http://www.oxymoronlist.com/), but it is essentially self-contradictory and not technically correct. As Ethan said, vitrify means turn into glass. Now, a glass state is a disordered solid state by definition, then it can't be a crystal. A vitrified crystal would be a crystal which has lost all three-dimensional ordering, pretty much like the material one gets when using the wrong cryo-protectant. What one usually does is to soak the crystal in a cryo-protectant and then flash-freeze the resulting material, hoping that the crystal structure will be preserved, while the rest remains disordered in a solid state (vitrified), so that it won't produce a diffraction pattern by itself, and will hold the crystal in a fixed position (very convenient for data collection). Moreover, I would say that clarifying a material is vitrified when subjected to liquid N2 temperatures would be required only if you were working with some liquid solvent which might remain in the liquid phase at that temperature, instead of the usual solid disordered state, but this is never the case with protein crystals. So, I vote for frozen crystal.- Javier PS: that comment by James Stroud I forgot to mention that if any dictionary is an authority on the very cold, it would be the Penguin dictionary., is hilarious, we need a Like button in the CCP4bb list! -- Javier M. Gonzalez Protein Crystallography Station Bioscience Division Los Alamos National Laboratory TA-43, Building 1, Room 172-G Mailstop M888 Phone: (505) 667-9376 On Thu, Nov 15, 2012 at 2:24 PM, Craig Bingman cbing...@biochem.wisc.eduwrote: cryopreserved It says that the crystals were transferred to cryogenic temperatures in an attempt to increase their lifetime in the beam, and avoids all of the other problems with all of the other language described. I was really trying to stay out of this, because I understand what everyone means with all of their other word choices. On Nov 15, 2012, at 2:07 PM, James Stroud wrote: Isn't cryo-cooled redundant? James On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote: Perhaps it's an artisan organic locavore fruit cake. Either way, your *crystal* is not vitrified. The solvent in your crystal might be glassy but your protein better still hold crystalline order (cf. ice) or you've wasted your time. Ergo, cryo-cooled is the description to use. Phil Jeffrey Princeton On 11/15/12 1:14 PM, Nukri Sanishvili wrote: s: An alternative way to avoid the argument and discussion all together is to use cryo-cooled. Tim: You go to a restaurant, spend all that time and money and order a fruitcake? Cheers, N.
