Re: [ccp4bb] offtopic__which crystals to harvest
Actually, UV fluorescence imaging appears to be a pretty reliable means of discriminating protein crystals, provided your protein has Trp residue(s). V. Nagarajan JAN Scientific, Inc. http://www.janscientific.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Artem Evdokimov Sent: Tuesday, January 13, 2009 8:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] offtopic__which crystals to harvest Hi, A few simple hints: (Please note that I am aware of the inexact language in the statements below but I don't have the time to write this up exactly - conversational English would have to do. Caveat emptor.) Most protein crystals will break or deform when poked with a steel needle. Most inorganic salts/minerals won't deform from a simple poke, but will break (often with an audible crack) when pushed hard against something solid (like the surface of the glass slip etc.). Many, but not all protein crystals can survive gentle prodding with a thin cat whisker. Nylon loops are a bit trickier because they can exhert different forces depending on their geometry, age of the loop, and user's manual aptitude. To make matters more complicated - crystals of organic materials (i.e. not salt but also not protein) can display properties similar to either (but will more often than not tend to behave like salts). Salt crystals sink very rapidly in most well solutions. Protein crystals often take their time (lower density). Salt crystals often display Newton rings (Newton rainbows) when viewed through a polarizer-analyzer pair (not to be confused with relatively simple gradients of birefringence colors that are also common to protein crystals!). I have seen a few crystals of proteins that had distinct Newton rings and they were all exceptionally good diffractors. Don't be confused by rainbow-like coloring that's often associated with spherolites - the latter aren't likely to diffract X-rays in a useful manner :-) If in doubt - stick your crystals into an X-ray beam. Pretty much the best way to resolve this ambiguity! The next best choice is to show the crystals to an experienced crystallographer - oftentimes it's possible to guess just by eyeballing the drops but it takes experience to learn the traits and habits. Membrane crystals (or for that matter any crystals grown in the presence of detergent) can be extremely tricky to identify correctly due to the inherently soft and nasty nature of detergent crystals and the tendency of the latter to form various quasi-crystalline artefacts. Good luck, Artem From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of deliang Sent: Tuesday, January 13, 2009 10:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] offtopic__which crystals to harvest Hi there, Since a lot of different forms of crystals shows, I am using a quick/simple strategy to choose crystals by applying a force on the crystal against the wall, with the nylon loop. Some can never break apart, so they are salt crystals? The others can not survive the force and lose their intact shape and sharp surface. It seems these are protein crystals, but are they bad crystals? I just came to this field, and welcome all your suggestions and experience. Thanks a lot. Deliang
Re: [ccp4bb] offtopic__which crystals to harvest
True. It also appears that most people will be looking for less expensive ways to distinguish between protein and salt crystals Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine From: CCP4 bulletin board on behalf of V. Nagarajan Sent: Wed 1/14/2009 2:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] offtopic__which crystals to harvest Actually, UV fluorescence imaging appears to be a pretty reliable means of discriminating protein crystals, provided your protein has Trp residue(s). V. Nagarajan JAN Scientific, Inc. http://www.janscientific.com http://www.janscientific.com/ -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Artem Evdokimov Sent: Tuesday, January 13, 2009 8:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] offtopic__which crystals to harvest Hi, A few simple hints: (Please note that I am aware of the inexact language in the statements below but I don't have the time to write this up exactly - conversational English would have to do. Caveat emptor.) Most protein crystals will break or deform when poked with a steel needle. Most inorganic salts/minerals won't deform from a simple poke, but will break (often with an audible crack) when pushed hard against something solid (like the surface of the glass slip etc.). Many, but not all protein crystals can survive gentle prodding with a thin cat whisker. Nylon loops are a bit trickier because they can exhert different forces depending on their geometry, age of the loop, and user's manual aptitude. To make matters more complicated - crystals of organic materials (i.e. not salt but also not protein) can display properties similar to either (but will more often than not tend to behave like salts). Salt crystals sink very rapidly in most well solutions. Protein crystals often take their time (lower density). Salt crystals often display Newton rings (Newton rainbows) when viewed through a polarizer-analyzer pair (not to be confused with relatively simple gradients of birefringence colors that are also common to protein crystals!). I have seen a few crystals of proteins that had distinct Newton rings and they were all exceptionally good diffractors. Don't be confused by rainbow-like coloring that's often associated with spherolites - the latter aren't likely to diffract X-rays in a useful manner :-) If in doubt - stick your crystals into an X-ray beam. Pretty much the best way to resolve this ambiguity! The next best choice is to show the crystals to an experienced crystallographer - oftentimes it's possible to guess just by eyeballing the drops but it takes experience to learn the traits and habits. Membrane crystals (or for that matter any crystals grown in the presence of detergent) can be extremely tricky to identify correctly due to the inherently soft and nasty nature of detergent crystals and the tendency of the latter to form various quasi-crystalline artefacts. Good luck, Artem From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of deliang Sent: Tuesday, January 13, 2009 10:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] offtopic__which crystals to harvest Hi there, Since a lot of different forms of crystals shows, I am using a quick/simple strategy to choose crystals by applying a force on the crystal against the wall, with the nylon loop. Some can never break apart, so they are salt crystals? The others can not survive the force and lose their intact shape and sharp surface. It seems these are protein crystals, but are they bad crystals? I just came to this field, and welcome all your suggestions and experience. Thanks a lot. Deliang
[ccp4bb] offtopic__which crystals to harvest
Hi there, Since a lot of different forms of crystals shows, I am using a quick/simple strategy to choose crystals by applying a force on the crystal against the wall, with the nylon loop. Some can never break apart, so they are salt crystals? The others can not survive the force and lose their intact shape and sharp surface. It seems these are protein crystals, but are they bad crystals? I just came to this field, and welcome all your suggestions and experience. Thanks a lot. Deliang
Re: [ccp4bb] offtopic__which crystals to harvest
Hi, A few simple hints: (Please note that I am aware of the inexact language in the statements below but I don't have the time to write this up exactly - conversational English would have to do. Caveat emptor.) Most protein crystals will break or deform when poked with a steel needle. Most inorganic salts/minerals won't deform from a simple poke, but will break (often with an audible crack) when pushed hard against something solid (like the surface of the glass slip etc.). Many, but not all protein crystals can survive gentle prodding with a thin cat whisker. Nylon loops are a bit trickier because they can exhert different forces depending on their geometry, age of the loop, and user's manual aptitude. To make matters more complicated - crystals of organic materials (i.e. not salt but also not protein) can display properties similar to either (but will more often than not tend to behave like salts). Salt crystals sink very rapidly in most well solutions. Protein crystals often take their time (lower density). Salt crystals often display Newton rings (Newton rainbows) when viewed through a polarizer-analyzer pair (not to be confused with relatively simple gradients of birefringence colors that are also common to protein crystals!). I have seen a few crystals of proteins that had distinct Newton rings and they were all exceptionally good diffractors. Don't be confused by rainbow-like coloring that's often associated with spherolites - the latter aren't likely to diffract X-rays in a useful manner :-) If in doubt - stick your crystals into an X-ray beam. Pretty much the best way to resolve this ambiguity! The next best choice is to show the crystals to an experienced crystallographer - oftentimes it's possible to guess just by eyeballing the drops but it takes experience to learn the traits and habits. Membrane crystals (or for that matter any crystals grown in the presence of detergent) can be extremely tricky to identify correctly due to the inherently soft and nasty nature of detergent crystals and the tendency of the latter to form various quasi-crystalline artefacts. Good luck, Artem _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of deliang Sent: Tuesday, January 13, 2009 10:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] offtopic__which crystals to harvest Hi there, Since a lot of different forms of crystals shows, I am using a quick/simple strategy to choose crystals by applying a force on the crystal against the wall, with the nylon loop. Some can never break apart, so they are salt crystals? The others can not survive the force and lose their intact shape and sharp surface. It seems these are protein crystals, but are they bad crystals? I just came to this field, and welcome all your suggestions and experience. Thanks a lot. Deliang
Re: [ccp4bb] offtopic__which crystals to harvest
1. Pick many representative samples from each morphological class. 2. Take as many notes about the appearance, habits, and physical properties of each crystal that you can. 3. Screen every crystal for diffraction in a variety of cryo- protectants, etc. 4. All protein crystals were meant to be destroyed in an x-ray beam pointed at a detector. 5. Salt has a characteristic diffraction. X-ray is the best way to test for salt. 6. If you can unambiguously identify salt crystals from the information you gathered in 2, 3, and 5, don't waste time harvesting said salt crystals. 7. Go to 1 until you have exhausted your supply of protein crystals. 8. Grow new crystals by varying growth conditions. 9. Go to 1 until you are happy with your data. James On Jan 13, 2009, at 7:50 PM, deliang wrote: Hi there, Since a lot of different forms of crystals shows, I am using a quick/ simple strategy to choose crystals by applying a force on the crystal against the wall, with the nylon loop. Some can never break apart, so they are salt crystals? The others can not survive the force and lose their intact shape and sharp surface. It seems these are protein crystals, but are they bad crystals? I just came to this field, and welcome all your suggestions and experience. Thanks a lot. Deliang
