[ccp4bb] AW: [ccp4bb] Detergent/lipid crystal diffraction pattern?
Hi Pius DS_1.png - protein diffraction ! DSA6E.png - surface ice formation and protein crystal which did not survive the freezing !! DSA2F.png - protein crystal which does not diffract, but cryo condition is right ! Psp4f.png - internal ice formation plus some surface ice also protein crystal Cheers Stefan
Re: [ccp4bb] Detergent/lipid crystal diffraction pattern?
Pius, Are you sure that you determined the correct cell. Which program did you use? Usually there are much less spots on an image when a crystal has so small unit size dimensions. To me the first crystal looks like protein. Send it to a synchrotron and process the data in XDS. They can process for you. Maia On 21/03/2011 4:33 PM, Maia Cherney wrote: Hi PS What is the unit cell dimensions in the first crystal? It looks like protein to me. Maia On 21/03/2011 2:03 PM, Pius Padayatti wrote: Hi all, We recently observed some diffraction from membrane protein crystallization drops diffraction that look like non-proteinaceous (please see attached files, from 4 different crystals grown in different conditions). Rains' question about about lipid and detergent diffraction is so relevant. This is most likely what lipids and detergent diffraction looks like? People with similar experience and know what could be these patterns might be from may have better suggestions and would like to hear all comments. first four images are from drops where detergent is DDM and and vapor diffusion while last image is from a crystal grown in mesophase (with monoolein). Padayatti PS On Sat, Mar 19, 2011 at 7:19 PM,Rain Field rainfiel...@163.com wrote: Hi All, I am wondering if the detergent or lipid crystal can have diffraction at low resolution. If they can, what does the diffraction pattern looks like? Are there any literatures describing these? Many thanks!
[ccp4bb] S-200 buffer-based peak shift?
Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] S-200 buffer-based peak shift?
At 07:23 PM 3/22/2011, Jacob Keller wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) So, it elutes earlier in essentially zero salt. I would bet that the protein is acidic and what you see is a buffer effect. Superdex (and most other gel filtration matrices) carries residual negative charge. So in zero salt there will be repulsion between protein and beads, resulting in the protein entering pore less frequently. Hence the earlier elution. I've seen this effect for a couple of monomeric acidic proteins. Chances are, switching to a salt higher than 50 mM will also retard the elution a bit. Typical recommended salt in gel filtration is in 100-200 mM range precisely to suppress ionic interactions. - Dima
Re: [ccp4bb] S-200 buffer-based peak shift?
I was always told that gel filtration resins have a mild ion-exchange character, hence the recommendation to use at least 100mM NaCl in size exclusion buffers. Assuming that it is true, one would expect a protein to stick to the resin in low salt buffers. That is the opposite of what you see (your protein elutes earlier in low salt buffer). That makes me think that you are actually experiencing some sort of oligomerization and/or shape transition. Do you have access to MALS? It would give you a definitive answer. On Tue, Mar 22, 2011 at 8:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] S-200 buffer-based peak shift?
Jacob, Some protein can form weak dimer, especially in low salt buffer. AUC can provide a more detailed info about your protein dimerization state. Ray On Mar 22, 2011, at 8:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] S-200 buffer-based peak shift?
Superdex 200 instruction manual suggests a minimal 150mM NaCl is required to prevent binding of protein to the resin. But it seems more to the side of preventing loss of protein instead of misjudging protein size. Nian Huang, Ph.D. UT Southwestern Medical Center On Tue, Mar 22, 2011 at 7:23 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] while on the subject of stereo
My 2 cents worth on the stereo-dependent: 1) They have carpal tunnel syndrome that makes it painful to keep the molecule in motion while rebuilding it (NOTE: enough constant mouse-wiggling and you will get carpal tunnel problems if you don't have them yet!) 2) They work on big, low-resolution structures where you need to see a bigger-picture view. I've had people tell me that can fit 3-3.5A maps just fine without stereo, but having viewed their work, I beg to differ. Phoebe Original message Date: Tue, 1 Mar 2011 22:30:54 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jan Löwe j...@mrc-lmb.cam.ac.uk) Subject: Re: [ccp4bb] while on the subject of stereo To: CCP4BB@JISCMAIL.AC.UK Ah! The question of to stereo or not to stereo! There has to be a scientific reason why this question is more popular than asking for what Linux distro is more fashionable this spring or why an Rmerge of 0.90 in the outermost shell is good for you and your structure. I am offering my two (conflicting) theories (and apologies that both seem to imply some problem): A) people who do use stereo have a problem with their brain because they cannot produce three dimensional vision from depth cues alone. B) people who do not use stereo have a problem with their brain because they cannot see properly in three dimensions and rely on depth cues alone. I personally prefer people with A) when I am their passenger in a car since they do not need to rotate by 90° to see how far the braking lights of the car in front are away :-) jan On 01/03/2011 21:35, Jim Pflugrath wrote: I will offer my view. I hate stereo glasses and hate stereo in general. One should be able to see 3D from the depth-cueing and by keeping the view in motion. For fitting, I like to flip the view by 90 degrees. I know I am going to move in displayX and displayY, but never in displayZ. I then rotate the view around the vertical axis so thatn the old displayZ becomes displayX. Furthermore, I don't waste too much time fitting. I know the software can fit the map better than me, so I let it do its job. I only need to get the coordinates within the radius of convergence of the refinement program. I also know that 9 times out of 10, the displayed electron density is probably suspect, so I believe in stereochemistry more than I believe in the map. The main trick is to realize that as a human being, you really are not that good at fitting the map or that it is unnecessary to waste your time since the software is really so much better than you. Refinement is quick enough that you can try various hypotheses as in: If I move this here, then refinement will do the trick and Well, that didn't work, so I will move that over there and see if refinement will do the trick. As for stereo figures, you should be able to convey what you want to say from a good figure with depth-cueing, shadows, etc. Don't ever use stereo glasses in a public seminar. Maybe my opinion will change with better stereo technology. OK, I know quite a lot of people will disagree with me. :) Jim -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Roberts Sent: Tuesday, March 01, 2011 10:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] while on the subject of stereo Hi again, I'd like to ask a question about the pedagogy of stereo. That is, using stereo with students in the classroom. Do you all find that, after setting up these elaborate stereo devices, students really use the stereo or do they tend not to? I am a huge fan of stereo - and frankly here we have quite a few options for doing stereo - from the active Nvidia systems that people have recently been discussing to passive zalmans. ... As I mentioned, I like stereo a lot, but really projecting on a nice bright lcd monitor also has it's advantages, and with the ease of moving things using the mouse (or whatever device you use), the overall need for stereo seems to be decreasing. I don't know - I just wonder what peoples views are out there for the actual need for stereo. It's incredibly cool - and I think is a very powerful way to show things - but I'm wondering if we focus too much on it because it's cool and not because it's pedagogically necessary. Just wondering, no worries. Thanks Dave
Re: [ccp4bb] while on the subject of stereo
what about the fashion statement made by cool glasses? From: Phoebe Rice [pr...@uchicago.edu] Sent: Tuesday, March 22, 2011 10:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] while on the subject of stereo My 2 cents worth on the stereo-dependent: 1) They have carpal tunnel syndrome that makes it painful to keep the molecule in motion while rebuilding it (NOTE: enough constant mouse-wiggling and you will get carpal tunnel problems if you don't have them yet!) 2) They work on big, low-resolution structures where you need to see a bigger-picture view. I've had people tell me that can fit 3-3.5A maps just fine without stereo, but having viewed their work, I beg to differ. Phoebe Original message Date: Tue, 1 Mar 2011 22:30:54 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jan Löwe j...@mrc-lmb.cam.ac.uk) Subject: Re: [ccp4bb] while on the subject of stereo To: CCP4BB@JISCMAIL.AC.UK Ah! The question of to stereo or not to stereo! There has to be a scientific reason why this question is more popular than asking for what Linux distro is more fashionable this spring or why an Rmerge of 0.90 in the outermost shell is good for you and your structure. I am offering my two (conflicting) theories (and apologies that both seem to imply some problem): A) people who do use stereo have a problem with their brain because they cannot produce three dimensional vision from depth cues alone. B) people who do not use stereo have a problem with their brain because they cannot see properly in three dimensions and rely on depth cues alone. I personally prefer people with A) when I am their passenger in a car since they do not need to rotate by 90° to see how far the braking lights of the car in front are away :-) jan On 01/03/2011 21:35, Jim Pflugrath wrote: I will offer my view. I hate stereo glasses and hate stereo in general. One should be able to see 3D from the depth-cueing and by keeping the view in motion. For fitting, I like to flip the view by 90 degrees. I know I am going to move in displayX and displayY, but never in displayZ. I then rotate the view around the vertical axis so thatn the old displayZ becomes displayX. Furthermore, I don't waste too much time fitting. I know the software can fit the map better than me, so I let it do its job. I only need to get the coordinates within the radius of convergence of the refinement program. I also know that 9 times out of 10, the displayed electron density is probably suspect, so I believe in stereochemistry more than I believe in the map. The main trick is to realize that as a human being, you really are not that good at fitting the map or that it is unnecessary to waste your time since the software is really so much better than you. Refinement is quick enough that you can try various hypotheses as in: If I move this here, then refinement will do the trick and Well, that didn't work, so I will move that over there and see if refinement will do the trick. As for stereo figures, you should be able to convey what you want to say from a good figure with depth-cueing, shadows, etc. Don't ever use stereo glasses in a public seminar. Maybe my opinion will change with better stereo technology. OK, I know quite a lot of people will disagree with me. :) Jim -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Roberts Sent: Tuesday, March 01, 2011 10:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] while on the subject of stereo Hi again, I'd like to ask a question about the pedagogy of stereo. That is, using stereo with students in the classroom. Do you all find that, after setting up these elaborate stereo devices, students really use the stereo or do they tend not to? I am a huge fan of stereo - and frankly here we have quite a few options for doing stereo - from the active Nvidia systems that people have recently been discussing to passive zalmans. ... As I mentioned, I like stereo a lot, but really projecting on a nice bright lcd monitor also has it's advantages, and with the ease of moving things using the mouse (or whatever device you use), the overall need for stereo seems to be decreasing. I don't know - I just wonder what peoples views are out there for the actual need for stereo. It's incredibly cool - and I think is a very powerful way to show things - but I'm wondering if we focus too much on it because it's cool and not because it's pedagogically necessary. Just wondering, no worries. Thanks Dave
Re: [ccp4bb] while on the subject of stereo
There are actually a bunch of depth cues that humans use to perceive 3-dimensionality. Existing computer displays only reproduce a few. Because of redundancy, people can function with only a subset, but it can be a serious handicap. Individuals who are stereoblind can learn to judge distances (such as how far away a stop sign is) from the sizes of familiar objects and other cues ... but it doesn't work that well. Conflicting depth cues are one thing that causes motion sickness. Rocking or rotating an image is a type of depth cue. Perspective is a depth cue. Lighting effects, such as fading the brightness of an object with distance or placing an object in fog are also depth cues. Focus is a depth due, and so is convergence of the eyes ... neither of those are reproduced by current computer graphics displays (except perhaps experimental ones). There is more to that list. A fair number of people have poor stereo vision ... some don't even realize it. It does not help that stereo systems are sometimes poorly adjusted and sometimes not even done mathematically correctly. Proper stereo transformations require that you know where the person's eyes are located with respect to the screen. A number of years ago, when I ran Cornell's virtual reality CAVE, one visitor told me he was stereo blind and so would not get much out of the experience. But something very unusual happened: when he put on the glasses and motion tracking system, he was apparently able to see stereo for the first time. Perhaps it was something to do with some misalignment in our system that compensated for his vision problem. Anyhow, it was a remarkable experience for him. I've fit density with and without stereo, and personally, I find that stereo adds a great deal. It's not everything, but why throw out a perfectly good depth cue and work stereoblind? It will be interesting to see if the current trend in stereo movies and 3D TV continues or fizzles (as it did in the 50's). Richard Gillilan MacCHESS On Mar 22, 2011, at 11:01 PM, Mayer, Mark (NIH/NICHD) [E] wrote: what about the fashion statement made by cool glasses? From: Phoebe Rice [pr...@uchicago.edu] Sent: Tuesday, March 22, 2011 10:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] while on the subject of stereo My 2 cents worth on the stereo-dependent: 1) They have carpal tunnel syndrome that makes it painful to keep the molecule in motion while rebuilding it (NOTE: enough constant mouse-wiggling and you will get carpal tunnel problems if you don't have them yet!) 2) They work on big, low-resolution structures where you need to see a bigger-picture view. I've had people tell me that can fit 3-3.5A maps just fine without stereo, but having viewed their work, I beg to differ. Phoebe Original message Date: Tue, 1 Mar 2011 22:30:54 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jan Löwe j...@mrc-lmb.cam.ac.uk) Subject: Re: [ccp4bb] while on the subject of stereo To: CCP4BB@JISCMAIL.AC.UK Ah! The question of to stereo or not to stereo! There has to be a scientific reason why this question is more popular than asking for what Linux distro is more fashionable this spring or why an Rmerge of 0.90 in the outermost shell is good for you and your structure. I am offering my two (conflicting) theories (and apologies that both seem to imply some problem): A) people who do use stereo have a problem with their brain because they cannot produce three dimensional vision from depth cues alone. B) people who do not use stereo have a problem with their brain because they cannot see properly in three dimensions and rely on depth cues alone. I personally prefer people with A) when I am their passenger in a car since they do not need to rotate by 90° to see how far the braking lights of the car in front are away :-) jan On 01/03/2011 21:35, Jim Pflugrath wrote: I will offer my view. I hate stereo glasses and hate stereo in general. One should be able to see 3D from the depth-cueing and by keeping the view in motion. For fitting, I like to flip the view by 90 degrees. I know I am going to move in displayX and displayY, but never in displayZ. I then rotate the view around the vertical axis so thatn the old displayZ becomes displayX. Furthermore, I don't waste too much time fitting. I know the software can fit the map better than me, so I let it do its job. I only need to get the coordinates within the radius of convergence of the refinement program. I also know that 9 times out of 10, the displayed electron density is probably suspect, so I believe in stereochemistry more than I believe in the map. The main trick is to realize that as a human being, you really are not that good at fitting the map or that it is unnecessary to waste your time since the software is really so much better than you.
