[ccp4bb] How to improve crystal which is twinning?
Hi, I have a 22kDa protein that the floopy N & C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng _ Windows Live™: Keep your life in sync. http://windowslive.com/explore?ocid=TXT_TAGLM_BR_life_in_synch_052009
Re: [ccp4bb] How to improve crystal which is twinning?
Dear Heng, I assume you are growing your crystals using vapor diffusion. If your optimization efforts using various additives fail to give you suitable crystals you might consider trying to grow your crystals using a different technique. I would suggest a liquid diffusion based technique such as 1) the Granada box from Triana <http://www.trianatech.com/Eng/Home.htm> http://www.trianatech.com/Eng/Home.htm or 2) the Crystal Former from Microlytic <http://www.microlytic.com> www.microlytic.com. Several of our (Microlytic's) customers have been able to convert poor crystallization hits from vapor diffusion to well diffracting crystals using the Crystal Former. The liquid diffusion mixing kinetics allow a unique sampling of protein phase space compared to vapor diffusion techniques, and can consequently be considered an orthogonal crystallization tool. This may be what you need to grow well ordered crystals of your protein. - Morten From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of HanJie_HCT Tai Sent: Sunday, May 17, 2009 8:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to improve crystal which is twinning? Hi, I have a 22kDa protein that the floopy N & C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng _ Windows LiveT: Keep your life in sync. Check it out. <http://windowslive.com/explore?ocid=TXT_TAGLM_BR_life_in_synch_052009>
Re: [ccp4bb] How to improve crystal which is twinning?
Try things that affect nucleation and kinetics of crystallization, such as seeding or crystallization at different temperatures. As Morten suggested, try different crystallization methods such as switching between hanging/sitting drop and microbatch. Screen protein concentration and protein:crystallization solution ratios carefully. Good luck! Ho UC Berkeley
Re: [ccp4bb] How to improve crystal which is twinning?
Heng-- Two things you might want to try: 1. Drop ratios with your current conditions and DMSO additive. Use different concentrations of DMSO too. 2. Try using Hampton Crystal Screen and Crystal Screen 2 as additives. I generally do this by adding 5% of XS 1 & 2 to the wells. Once I find one of these reagents that improves crystal quality/diffraction, I optimize the concentration for that. You can do this with the Mosquito robot for 96 well formator in the 24 well VDX trays. Hope this helps! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com "HanJie_HCT Tai" Sent by: "CCP4 bulletin board" 17-May-2009 08:27 Please respond to "HanJie_HCT Tai" To CCP4BB@JISCMAIL.AC.UK cc Subject [ccp4bb] How to improve crystal which is twinning? Hi, I have a 22kDa protein that the floopy N & C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng Windows Live?: Keep your life in sync. Check it out.
Re: [ccp4bb] How to improve crystal which is twinning?
Hello Annie, How effective have you found using sparse matrix screens as additives vs. traditional additive screens? I've tried this only a few times without success and would like to hear from someone with more experience. Thanks! Ho UC Berkeley -- Date:Mon, 18 May 2009 09:34:12 -0400 From:Annie Hassell Subject: Re: How to improve crystal which is twinning? Heng-- Two things you might want to try: 1. Drop ratios with your current conditions and DMSO additive. Use=20 different concentrations of DMSO too.=20 2. Try using Hampton Crystal Screen and Crystal Screen 2 as additives. I = generally do this by adding 5% of XS 1 & 2 to the wells. Once I find one=20 of these reagents that improves crystal quality/diffraction, I optimize=20 the concentration for that. You can do this with the Mosquito robot for=20 96 well formator in the 24 well VDX trays.=20 Hope this helps! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com
Re: [ccp4bb] How to improve crystal which is twinning?
Hi Ho-- We have had lots of success using the Hampton Research Crystal Screens 1 & 2 as additives. Sometimes these improve the crystal quality whereas the 96 Additive Screen does not. Whenever I screen for additives, I include Crystal Screen 1 & 2. What I have NOT had a great deal of success with is determining which component of the Crystal Screen reagent is responsible for improving the crystal quality. Does not appear that it is just one component in many cases. I generally use 5% of the sparse matrix screen added to the well. When I get a hit, then I further optimize the concentration of the reagent to add to the well. HTH! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com "Ho-Leung Ng" Sent by: holeung...@gmail.com 20-May-2009 18:39 To "CCP4 bulletin board" , annie.m.hass...@gsk.com cc Subject Re: How to improve crystal which is twinning? Hello Annie, How effective have you found using sparse matrix screens as additives vs. traditional additive screens? I've tried this only a few times without success and would like to hear from someone with more experience. Thanks! Ho UC Berkeley -- Date:Mon, 18 May 2009 09:34:12 -0400 From:Annie Hassell Subject: Re: How to improve crystal which is twinning? Heng-- Two things you might want to try: 1. Drop ratios with your current conditions and DMSO additive. Use=20 different concentrations of DMSO too.=20 2. Try using Hampton Crystal Screen and Crystal Screen 2 as additives. I = generally do this by adding 5% of XS 1 & 2 to the wells. Once I find one=20 of these reagents that improves crystal quality/diffraction, I optimize=20 the concentration for that. You can do this with the Mosquito robot for=20 96 well formator in the 24 well VDX trays.=20 Hope this helps! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com
Re: [ccp4bb] How to improve crystal which is twinning?
If you haven't already used it, I would certainly try the approach of microseeding into screens since you already have crystals http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 -- patr...@douglas.co.uk <mailto:patr...@douglas.co.uk> Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ <http://www.douglas.co.uk/> Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of HanJie_HCT Tai Sent: 17 May 2009 13:27 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to improve crystal which is twinning? Hi, I have a 22kDa protein that the floopy N & C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng Windows Live(tm): Keep your life in sync. Check it out. <http://windowslive.com/explore?ocid=TXT_TAGLM_BR_life_in_synch_052009>
