[ccp4bb] Fwd: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Dear Jacob, As mentioned before by Frank, many insect viruses use in vivo crystals as their main infectious form: *The molecular organization of cypovirus polyhedra.* Nature 446, 97–101 (2007). Coulibaly, F. et al. *The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.* Proc Natl Acad Sci USA 106, 22205–22210 (2009). Coulibaly, F. et al. You may also want to check out a short review if you are interested in more natural examples of in vivo crystallization: *Protein crystallization in vivo.* Curr Opin Colloid Interf Sci 11, 40–46 (2006). Doye, J. Poon, W. You can even see them forming in infected cells in the following video: http://www.jbc.org/content/suppl/2001/05/11/276.20.16704.DC1/Video3.mov Best, Fasseli On 16 February 2013 06:44, Jacob Keller j-kell...@fsm.northwestern.eduwrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Hello, I've seen similar crystals in HEK cells when expressing certain (but not most) mVenus-fusion proteins. mVenus is similar to mYFP. I now wonder if this effect depends on my protein being cleaved, releasing mVenus. Will look into this. Elena Original message Date: Sat, 16 Feb 2013 16:14:58 -0600 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Artem Evdokimov artem.evdoki...@gmail.com) Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! To: CCP4BB@JISCMAIL.AC.UK Note: the crystals in my paper are not regular GFP, they're special GFP from a marine Copepod. I've seen GFP crystals (eGFP) before in other cells, though. Bacillus thuringiensis (an organism I work with every day) makes beautiful crystals of parasporal insecticidal toxins when it sporulates. Hundreds of these proteins are known, and many of them (about 60% or so) produce geometrically perfect shapes (cubes, rectangles, etc.) The internet is rife with images of these if you care to see them. We are very interested in FEL studies of those crystals but we've not contacted the FEL folks yet (if any of them are reading, please feel free to comment back to me directly). This is probably the most abundant/diverse source of biologically crystallized protein material (at least as far as I know). Artem On Sat, Feb 16, 2013 at 4:04 PM, A. Radu Aricescu r...@strubi.ox.ac.uk wrote: Hi Artem, Jacob, I've personally never seen such crystals despite using for many years various stand-alone GFP variants (including eGFP) as transfection controls in HEKs and other mammalian cell lines, but now I believe you and owe Jacob an apology for being skeptical when I first saw his pics :-)) But, I can't stop wondering what is the secret? Must be something else than just overexpression... How often do you see such crystals occurring (what % of GFP-expressing cells), and how stable are they? Could this be related to the expression system, transfection procedure, broader cell culture conditions... Artem finds GFP crystals in HeLa cells (have you also tried HEKs?), while Jacob's experience seems to be the opposite (admittedly using a different GFP flavour)... It just feels like this in cell crystallization might become a very powerful tool if one could harness it! Best wishes, radu -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547 Original message Date: Sat, 16 Feb 2013 15:38:12 -0600 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Artem Evdokimov artem.evdoki...@gmail.com) Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! To: CCP4BB@JISCMAIL.AC.UK In addition to the above mentioned references you can also see: Charcot-Leyden crystals (http://www.ncbi.nlm.nih.gov/pubmed/6508005) and my own ( :) ) figure 1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618374/figure/f1/ Cheers, Artem On Fri, Feb 15, 2013 at 11:46 PM, Zhijie Li zhijie...@utoronto.ca wrote: Hi Jacob, Interesting topic. This reminds me the posters I saw on ACA 2010, on the femto-second infrared laser based instrument . That instrument utilizes the nonlinear optical properties of crystals of chiral molecules to detect very small crystalline materials from amorphous background: the crystals will double the frequency of the laser, turning the infrared light to visible light. I cannot recall the exact name of the technology now, unfortunately. Your case of observing in vivo GFP crystals is a little special in that the crystals are fluorescent. I guess if we scan cells over-expressing proteins with the above mentioned instrument, we might find that many proteins will do the same in cells. Naturally occurring in vivo crystals are not very rare. If we do not restrict the topic to proteins, then it is well known that many viruses readily crystallize in the host cell's nuclei and the resulting crystals or crystalline arrays can be observed under EM. And if we do not restrict the cells to mammalian cells, then there come the famous BT crystals
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Jacob, I recall Dorothy Hodgkin or Guy Dodson showing a slide (mid to late 1970s) with insulin crystals having grown in the Islets of Langerhans. As you say, quite remarkable. John Prof John R Helliwell DSc On 15 Feb 2013, at 19:44, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu *** GFP_crystals_DIC.png GFP_crystals_Fluorescence.png
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
On 02/16/13 00:46, Zhijie Li wrote: Hi Jacob, Interesting topic. This reminds me the posters I saw on ACA 2010, on the femto-second infrared laser based instrument . That instrument utilizes the nonlinear optical properties of crystals of chiral molecules to detect very small crystalline materials from amorphous background: the crystals will double the frequency of the laser, turning the infrared light to visible light. I cannot recall the exact name of the technology now, unfortunately. Multpile photon fluorescent microscopy, or two-photon excitation microscopy, sometimes implemented as scanning confocal microscopy. One implementation is acronymmed SONICC. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
In addition to the above mentioned references you can also see: Charcot-Leyden crystals (http://www.ncbi.nlm.nih.gov/pubmed/6508005) and my own ( :) ) figure 1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618374/figure/f1/ Cheers, Artem On Fri, Feb 15, 2013 at 11:46 PM, Zhijie Li zhijie...@utoronto.ca wrote: ** Hi Jacob, Interesting topic. This reminds me the posters I saw on ACA 2010, on the femto-second infrared laser based instrument . That instrument utilizes the nonlinear optical properties of crystals of chiral molecules to detect very small crystalline materials from amorphous background: the crystals will double the frequency of the laser, turning the infrared light to visible light. I cannot recall the exact name of the technology now, unfortunately. Your case of observing in vivo GFP crystals is a little special in that the crystals are fluorescent. I guess if we scan cells over-expressing proteins with the above mentioned instrument, we might find that many proteins will do the same in cells. Naturally occurring *in vivo *crystals are not very rare. If we do not restrict the topic to proteins, then it is well known that many viruses readily crystallize in the host cell's nuclei and the resulting crystals or crystalline arrays can be observed under EM. And if we do not restrict the cells to mammalian cells, then there come the famous BT crystals. In addition, I just did some internet search and here are some interesting results: 1) Viral protein crystals can form in HEK cells infected by adenovirus ( http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894) 2) Bacterial infection can cause the infected epithelial cells to form pathological crystal-containing inclusion bodies in the cytosol ( http://www.ncbi.nlm.nih.gov/pubmed/8940763). 3) Crystalline inclusion bodies are found in rabbit embryos ( http://dev.biologists.org/content/44/1/31.full.pdf) and epididymis of the nine-banded armadillo( http://www.sciencedirect.com/science/article/pii/S0022532073800073). Actually if google crystalline inclusion body, there will be tons of literatures. 4) IgG crystallized in the ER when over expressed from a highly optimized CHO expression system (http://www.jbc.org/content/286/22/19917.abstract). This is particularly interesting as we know that whole IgGs are not so prone to crystallize, although the author do state that Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG. Given the prevalence of *in vivo* crystallization, especially considering their correlation with inclusion bodies, I think it is reasonable to suspect that there are some cases that the inclusion bodies formed during over expression of transgenic proteins in *E. coli* are crystalline. I expect that we will be enlightened on this issue by somebody on the BB soon. Zhijie *From:* Jacob Keller j-kell...@fsm.northwestern.edu *Sent:* Friday, February 15, 2013 2:44 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Sighting of Protein Crystals in Vivo?! Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Note: the crystals in my paper are not regular GFP, they're special GFP from a marine Copepod. I've seen GFP crystals (eGFP) before in other cells, though. Bacillus thuringiensis (an organism I work with every day) makes beautiful crystals of parasporal insecticidal toxins when it sporulates. Hundreds of these proteins are known, and many of them (about 60% or so) produce geometrically perfect shapes (cubes, rectangles, etc.) The internet is rife with images of these if you care to see them. We are very interested in FEL studies of those crystals but we've not contacted the FEL folks yet (if any of them are reading, please feel free to comment back to me directly). This is probably the most abundant/diverse source of biologically crystallized protein material (at least as far as I know). Artem On Sat, Feb 16, 2013 at 4:04 PM, A. Radu Aricescu r...@strubi.ox.ac.ukwrote: Hi Artem, Jacob, I've personally never seen such crystals despite using for many years various stand-alone GFP variants (including eGFP) as transfection controls in HEKs and other mammalian cell lines, but now I believe you and owe Jacob an apology for being skeptical when I first saw his pics :-)) But, I can't stop wondering what is the secret? Must be something else than just overexpression... How often do you see such crystals occurring (what % of GFP-expressing cells), and how stable are they? Could this be related to the expression system, transfection procedure, broader cell culture conditions... Artem finds GFP crystals in HeLa cells (have you also tried HEKs?), while Jacob's experience seems to be the opposite (admittedly using a different GFP flavour)... It just feels like this in cell crystallization might become a very powerful tool if one could harness it! Best wishes, radu -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547 Original message Date: Sat, 16 Feb 2013 15:38:12 -0600 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Artem Evdokimov artem.evdoki...@gmail.com) Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! To: CCP4BB@JISCMAIL.AC.UK In addition to the above mentioned references you can also see: Charcot-Leyden crystals (http://www.ncbi.nlm.nih.gov/pubmed/6508005) and my own ( :) ) figure 1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618374/figure/f1/ Cheers, Artem On Fri, Feb 15, 2013 at 11:46 PM, Zhijie Li zhijie...@utoronto.ca wrote: Hi Jacob, Interesting topic. This reminds me the posters I saw on ACA 2010, on the femto-second infrared laser based instrument . That instrument utilizes the nonlinear optical properties of crystals of chiral molecules to detect very small crystalline materials from amorphous background: the crystals will double the frequency of the laser, turning the infrared light to visible light. I cannot recall the exact name of the technology now, unfortunately. Your case of observing in vivo GFP crystals is a little special in that the crystals are fluorescent. I guess if we scan cells over-expressing proteins with the above mentioned instrument, we might find that many proteins will do the same in cells. Naturally occurring in vivo crystals are not very rare. If we do not restrict the topic to proteins, then it is well known that many viruses readily crystallize in the host cell's nuclei and the resulting crystals or crystalline arrays can be observed under EM. And if we do not restrict the cells to mammalian cells, then there come the famous BT crystals. In addition, I just did some internet search and here are some interesting results: 1) Viral protein crystals can form in HEK cells infected by adenovirus ( http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894) 2) Bacterial infection can cause the infected epithelial cells to form pathological crystal-containing inclusion bodies in the cytosol (http://www.ncbi.nlm.nih.gov/pubmed/8940763). 3) Crystalline inclusion bodies are found in rabbit embryos (http://dev.biologists.org/content/44/1/31.full.pdf) and epididymis of the nine-banded armadillo( http://www.sciencedirect.com/science/article/pii/S0022532073800073). Actually if google crystalline inclusion body, there will be tons of literatures. 4) IgG crystallized in the ER when over expressed from a highly optimized CHO expression system (http://www.jbc.org/content/286/22/19917.abstract). This is particularly interesting as we know that whole IgGs
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Hi Jacob, check out Figure 1 in Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Redecke L, et al. Science. 2013 Jan 11;339(6116):227-30. and In vivo protein crystallization opens new routes in structural biology. Koopmann R. Nature Methods. 2012 Jan 29;9(3):259-62. best Savvas On 15 Feb 2013, at 20:44, Jacob Keller wrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu *** GFP_crystals_DIC.pngGFP_crystals_Fluorescence.png
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Of course, common in baculo: http://dx.doi.org/10.1038/emboj.2009.352 The EMBO Journal (2010) 29,505--514 *How baculovirus polyhedra fit square pegs into round holes to robustly package viruses* Xiaoyun Ji, Geoff Sutton, Gwyndaf Evans, Danny Axford, Robin Owen and David I Stuart On 15/02/2013 20:00, Savvas Savvides wrote: Hi Jacob, check out Figure 1 in Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Redecke L, et al. Science. 2013 Jan 11;339(6116):227-30. and In vivo protein crystallization opens new routes in structural biology. Koopmann R. Nature Methods. 2012 Jan 29;9(3):259-62. best Savvas On 15 Feb 2013, at 20:44, Jacob Keller wrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu *** GFP_crystals_DIC.pngGFP_crystals_Fluorescence.png
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Hi Jacob, They are not too small to mount I think, at least 20 microns long (compared to the size of surrounding cells). But what do you mean by littered? There seems to be just one cell with crystals out of say 10 expressing eGFP. And why are there only 10 or so expressing GFP out of ~200 or more cells in the field? This does not look like transient expression using a normal plasmid... Very intriguing nevertheless! radu -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547 Original message Date: Fri, 15 Feb 2013 14:44:32 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jacob Keller j-kell...@fsm.northwestern.edu) Subject: [ccp4bb] Sighting of Protein Crystals in Vivo?! To: CCP4BB@JISCMAIL.AC.UK Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu *** GFP_crystals_DIC.png (938k bytes) GFP_crystals_Fluorescence.png (586k bytes)
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
My favorite: Hum Mol Genet. 2000 Jul 22;9(12):1779-86. Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novák P, Rezácová P, Ondrová L, Filipec M, Sedlácek J, Elleder M. http://hmg.oxfordjournals.org/content/9/12/1779.long From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, February 15, 2013 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! Of course, common in baculo: http://dx.doi.org/10.1038/emboj.2009.352 The EMBO Journal (2010) 29,505-514 How baculovirus polyhedra fit square pegs into round holes to robustly package viruses Xiaoyun Ji, Geoff Sutton, Gwyndaf Evans, Danny Axford, Robin Owen and David I Stuart On 15/02/2013 20:00, Savvas Savvides wrote: Hi Jacob, check out Figure 1 in Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Redecke L, et al. Science. 2013 Jan 11;339(6116):227-30. and In vivo protein crystallization opens new routes in structural biology. Koopmann R. Nature Methods. 2012 Jan 29;9(3):259-62. best Savvas On 15 Feb 2013, at 20:44, Jacob Keller wrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** GFP_crystals_DIC.pngGFP_crystals_Fluorescence.png
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
I am impressed that within a matter of hours, we now have a number of references for papers describing so-called in vivo crystallization. Wow, the benefits of a good network I guess. This kind of quick feedback would be fantastic for authors who are writing review articles... Dave Waugh On 2/15/13 3:27 PM, Michael Kothe michael.ko...@genzyme.com wrote: My favorite: Hum Mol Genet. 2000 Jul 22;9(12):1779-86. Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novák P, Rezácová P, Ondrová L, Filipec M, Sedlácek J, Elleder M. http://hmg.oxfordjournals.org/content/9/12/1779.long From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, February 15, 2013 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! Of course, common in baculo: http://dx.doi.org/10.1038/emboj.2009.352 The EMBO Journal (2010) 29,505–514 How baculovirus polyhedra fit square pegs into round holes to robustly package viruses Xiaoyun Ji, Geoff Sutton, Gwyndaf Evans, Danny Axford, Robin Owen and David I Stuart On 15/02/2013 20:00, Savvas Savvides wrote: Hi Jacob, check out Figure 1 in Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Redecke L, et al. Science. 2013 Jan 11;339(6116):227-30. and In vivo protein crystallization opens new routes in structural biology. Koopmann R. Nature Methods. 2012 Jan 29;9(3):259-62. best Savvas On 15 Feb 2013, at 20:44, Jacob Keller wrote: Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- David S. Waugh, Ph.D. Macromolecular Crystallography Laboratory Center for Cancer Research National Cancer Institute Bldg. 538, Room 209A Frederick, MD 21702-1201 +1 (301) 846-1842 wau...@mail.nih.gov http://mcl1.ncifcrf.gov/waugh.html --
Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
Hi Jacob, Interesting topic. This reminds me the posters I saw on ACA 2010, on the femto-second infrared laser based instrument . That instrument utilizes the nonlinear optical properties of crystals of chiral molecules to detect very small crystalline materials from amorphous background: the crystals will double the frequency of the laser, turning the infrared light to visible light. I cannot recall the exact name of the technology now, unfortunately. Your case of observing in vivo GFP crystals is a little special in that the crystals are fluorescent. I guess if we scan cells over-expressing proteins with the above mentioned instrument, we might find that many proteins will do the same in cells. Naturally occurring in vivo crystals are not very rare. If we do not restrict the topic to proteins, then it is well known that many viruses readily crystallize in the host cell's nuclei and the resulting crystals or crystalline arrays can be observed under EM. And if we do not restrict the cells to mammalian cells, then there come the famous BT crystals. In addition, I just did some internet search and here are some interesting results: 1) Viral protein crystals can form in HEK cells infected by adenovirus (http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894) 2) Bacterial infection can cause the infected epithelial cells to form pathological crystal-containing inclusion bodies in the cytosol (http://www.ncbi.nlm.nih.gov/pubmed/8940763). 3) Crystalline inclusion bodies are found in rabbit embryos (http://dev.biologists.org/content/44/1/31.full.pdf) and epididymis of the nine-banded armadillo(http://www.sciencedirect.com/science/article/pii/S0022532073800073). Actually if google crystalline inclusion body, there will be tons of literatures. 4) IgG crystallized in the ER when over expressed from a highly optimized CHO expression system (http://www.jbc.org/content/286/22/19917.abstract). This is particularly interesting as we know that whole IgGs are not so prone to crystallize, although the author do state that Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG. Given the prevalence of in vivo crystallization, especially considering their correlation with inclusion bodies, I think it is reasonable to suspect that there are some cases that the inclusion bodies formed during over expression of transgenic proteins in E. coli are crystalline. I expect that we will be enlightened on this issue by somebody on the BB soon. Zhijie From: Jacob Keller Sent: Friday, February 15, 2013 2:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Sighting of Protein Crystals in Vivo?! Dear Crystallographers, I was looking at some live, control HEK cells expressing just eGFP, and to my great surprise, saw littered across the dish what appeared to be small fluorescent needles (see attached--sorry about the size, but it's only ~1MB total.) Can these possibly be fortuitous protein crystals? They were too small to mount I think, and for what it's worth, parallel-transfected HeLa cells did not have these things. But, some needles could be seen in the DIC images as well, and the needles were only fluorescent with GFP filter sets, and not CFP, YFP, or texas red filters. I thought of whale myoglobin crystallizing on the decks of ships, but never thought I would see this Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
