[ccp4bb] fastening crystal formation
Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975
Re: [ccp4bb] fastening crystal formation
I recommend to try seeding! On 31.10.2014 14:05, Vijaykumar Pillalamarri wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975 -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] fastening crystal formation
Dear, the conditions you list should equilibrate within a couple of days. A growth time of three months could be a sign of some proteolytic reaction happening. You might want to sequence the crystal and subclone the fragment that actually crystallises. Best, Tim On 10/31/2014 11:05 AM, Vijaykumar Pillalamarri wrote: > Dear all, > > I am trying to crystallize a 30 kD protein. Protein crystals are formed > after 3 months. The crystals are formed in the following condition: > 0.01M Zinc sulphate > 0.1M MES monohydrate pH 6.5 > 25% v/v PEG monomethyl ether 550 > > Please suggest me how to grow these crystals faster. > > Thanking you > -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] fastening crystal formation
Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri wrote: > Dear all, > > I am trying to crystallize a 30 kD protein. Protein crystals are formed after > 3 months. The crystals are formed in the following condition: > 0.01M Zinc sulphate > 0.1M MES monohydrate pH 6.5 > 25% v/v PEG monomethyl ether 550 > > Please suggest me how to grow these crystals faster. > > Thanking you > > -- > Vijaykumar Pillalamarri, > UGC-JRF, > C/O: Dr. Anthony Addlagatta, > Senior Scientist, > CSIR-IICT, Tarnaka, > Hyderabad, India-57 > Mobile: +918886922975
Re: [ccp4bb] fastening crystal formation
I assume you were using vapour diffusion. In that case, you could also try microbatch to cover the possibility of the plate slowly drying out and concentrations increasing beyond those of the original screen. Dmitry On Fri, Oct 31, 2014 at 12:07 PM, R. M. Garavito wrote: > Although three months is a long time, it is no completely unheard of, and > does not require the invocation of proteolysis. The longest time I have > heard of is ~1 yr, so count yourself lucky. However to get good advice, as > well as to use it, you need to ask yourself several questions: > > 1. What kind of crystals are they? Protein, salt, etc? If they are salt, > don't pursue this condition. > > 2. How many crystals did you get? One or 2 in a drop or a microcrystal > shower. And of what kind? Single, well shaped, rosettes, needle clusters, > or something that looks crystalline. Screen more broadly around your > initial hit. > > 3. How many times have your tried to repeat this? Once, twice, or more? > Did you try setups in duplicate? If so, did you get reproducible results? > Have you actually screened around these conditions, varying each component > systematically (PEG, salt, pH, buffer, etc.)? > > 4. What method did you use? And in what kind of container? For one > thing, we don't completely trust the integrity of our setups for longer > than 2 months. All containers leak water slowly, so when crystals take > longer than 2 months to grow (a) the real conditions are at much higher > values than you naively think (i.e., the drop has dried out more than you > expected) or (b) other components are crystallizing, for example a zinc > salt. It depends what else is in your protein buffer, as well. > > To quicken protein crystallization (which is not always a good thing), > increase your protein concentration (by 1.5-2x) and/or PEG concentration > (such as screening up to 40% PEGmme 550). Sadly, crystallization is a > combination of thermodynamic and kinetic factors: you can get crystals > (sometimes a single crystal only) when just outside the truly optimal > conditions, but this may be only a sporadic event. You got to keep > screening. > > Good luck, > > Michael > > > ** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *603 Wilson Rd., Rm. 513* > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:* *(517) 355-9724 <%28517%29%20355-9724> Lab: (517) > 353-9125 <%28517%29%20353-9125>* > *FAX: (517) 353-9334 <%28517%29%20353-9334> > Email: rmgarav...@gmail.com * > ** > > > > > On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < > vijaypkuma...@gmail.com> wrote: > > Dear all, > > I am trying to crystallize a 30 kD protein. Protein crystals are formed > after 3 months. The crystals are formed in the following condition: > 0.01M Zinc sulphate > 0.1M MES monohydrate pH 6.5 > 25% v/v PEG monomethyl ether 550 > > Please suggest me how to grow these crystals faster. > > Thanking you > > -- > Vijaykumar Pillalamarri, > UGC-JRF, > C/O: Dr. Anthony Addlagatta, > Senior Scientist, > CSIR-IICT, Tarnaka, > Hyderabad, India-57 > Mobile: +918886922975 > > >
Re: [ccp4bb] fastening crystal formation
Michael (and some others) You haven't mentioned - and I guess don't use regularly - the "random" MMS approach, where crushed seed-crystals are added to random screens. This really is a terrific method, and it will on average roughly double productivity. It's the first thing I would think of in a case like Vijaykumar's (as I told him this morning!). Galina Obmolova and her colleagues published a great paper earlier this year about MMS. They were working with a set of 16 Fab fragments that comprised all combinations of 4 different heavy chains and 4 different light chains. Three structures were generated without MMS, but by various very creative uses of microseeding they were able to get all 16/16 structures. Ref below. Best wishes, Patrick __ Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., & Gilliland, G. L. (2014). Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. *Structural Biology and Crystallization Communications*, *70*(8). On 31 October 2014 16:07, R. M. Garavito wrote: > Although three months is a long time, it is no completely unheard of, and > does not require the invocation of proteolysis. The longest time I have > heard of is ~1 yr, so count yourself lucky. However to get good advice, as > well as to use it, you need to ask yourself several questions: > > 1. What kind of crystals are they? Protein, salt, etc? If they are salt, > don't pursue this condition. > > 2. How many crystals did you get? One or 2 in a drop or a microcrystal > shower. And of what kind? Single, well shaped, rosettes, needle clusters, > or something that looks crystalline. Screen more broadly around your > initial hit. > > 3. How many times have your tried to repeat this? Once, twice, or more? > Did you try setups in duplicate? If so, did you get reproducible results? > Have you actually screened around these conditions, varying each component > systematically (PEG, salt, pH, buffer, etc.)? > > 4. What method did you use? And in what kind of container? For one > thing, we don't completely trust the integrity of our setups for longer > than 2 months. All containers leak water slowly, so when crystals take > longer than 2 months to grow (a) the real conditions are at much higher > values than you naively think (i.e., the drop has dried out more than you > expected) or (b) other components are crystallizing, for example a zinc > salt. It depends what else is in your protein buffer, as well. > > To quicken protein crystallization (which is not always a good thing), > increase your protein concentration (by 1.5-2x) and/or PEG concentration > (such as screening up to 40% PEGmme 550). Sadly, crystallization is a > combination of thermodynamic and kinetic factors: you can get crystals > (sometimes a single crystal only) when just outside the truly optimal > conditions, but this may be only a sporadic event. You got to keep > screening. > > Good luck, > > Michael > > > ** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *603 Wilson Rd., Rm. 513* > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:* *(517) 355-9724 Lab: (517) 353-9125* > *FAX: (517) 353-9334Email: rmgarav...@gmail.com > * > ** > > > > > On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < > vijaypkuma...@gmail.com> wrote: > > Dear all, > > I am trying to crystallize a 30 kD protein. Protein crystals are formed > after 3 months. The crystals are formed in the following condition: > 0.01M Zinc sulphate > 0.1M MES monohydrate pH 6.5 > 25% v/v PEG monomethyl ether 550 > > Please suggest me how to grow these crystals faster. > > Thanking you > > -- > Vijaykumar Pillalamarri, > UGC-JRF, > C/O: Dr. Anthony Addlagatta, > Senior Scientist, > CSIR-IICT, Tarnaka, > Hyderabad, India-57 > Mobile: +918886922975 > > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart 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
Re: [ccp4bb] fastening crystal formation
Hello, If the homologous protein structure is available, or you have symmetry information of your crystal, then try engineering surface by putting in residues which has less entropy like replacing Lys to Arg, Met to Gln. We are able to fasten the crystal growth from 20 days to 3-4 days by creating phenylalanine Pi-stack. This is tricky but if you have an idea of crystal contact, then it should be straightforward without much trouble. Good Luck Appu On 31 October 2014 11:43, Patrick Shaw Stewart wrote: > > Michael (and some others) > > You haven't mentioned - and I guess don't use regularly - the "random" MMS > approach, where crushed seed-crystals are added to random screens. This > really is a terrific method, and it will on average roughly double > productivity. It's the first thing I would think of in a case like > Vijaykumar's (as I told him this morning!). > > Galina Obmolova and her colleagues published a great paper earlier this > year about MMS. They were working with a set of 16 Fab fragments that > comprised all combinations of 4 different heavy chains and 4 different > light chains. Three structures were generated without MMS, but by various > very creative uses of microseeding they were able to get all 16/16 > structures. Ref below. > > Best wishes, > > Patrick > > __ > > > Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., & Gilliland, G. > L. (2014). Protein crystallization with microseed matrix screening: > application to human germline antibody Fabs. *Structural Biology and > Crystallization Communications*, *70*(8). > > > > > On 31 October 2014 16:07, R. M. Garavito wrote: > >> Although three months is a long time, it is no completely unheard of, and >> does not require the invocation of proteolysis. The longest time I have >> heard of is ~1 yr, so count yourself lucky. However to get good advice, as >> well as to use it, you need to ask yourself several questions: >> >> 1. What kind of crystals are they? Protein, salt, etc? If they are >> salt, don't pursue this condition. >> >> 2. How many crystals did you get? One or 2 in a drop or a microcrystal >> shower. And of what kind? Single, well shaped, rosettes, needle clusters, >> or something that looks crystalline. Screen more broadly around your >> initial hit. >> >> 3. How many times have your tried to repeat this? Once, twice, or >> more? Did you try setups in duplicate? If so, did you get reproducible >> results? Have you actually screened around these conditions, varying each >> component systematically (PEG, salt, pH, buffer, etc.)? >> >> 4. What method did you use? And in what kind of container? For one >> thing, we don't completely trust the integrity of our setups for longer >> than 2 months. All containers leak water slowly, so when crystals take >> longer than 2 months to grow (a) the real conditions are at much higher >> values than you naively think (i.e., the drop has dried out more than you >> expected) or (b) other components are crystallizing, for example a zinc >> salt. It depends what else is in your protein buffer, as well. >> >> To quicken protein crystallization (which is not always a good thing), >> increase your protein concentration (by 1.5-2x) and/or PEG concentration >> (such as screening up to 40% PEGmme 550). Sadly, crystallization is a >> combination of thermodynamic and kinetic factors: you can get crystals >> (sometimes a single crystal only) when just outside the truly optimal >> conditions, but this may be only a sporadic event. You got to keep >> screening. >> >> Good luck, >> >> Michael >> >> >> ** >> *R. Michael Garavito, Ph.D.* >> *Professor of Biochemistry & Molecular Biology* >> *603 Wilson Rd., Rm. 513* >> *Michigan State University * >> *East Lansing, MI 48824-1319* >> *Office:* *(517) 355-9724 <%28517%29%20355-9724> Lab: (517) >> 353-9125 <%28517%29%20353-9125>* >> *FAX: (517) 353-9334 <%28517%29%20353-9334> >> Email: rmgarav...@gmail.com * >> ** >> >> >> >> >> On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < >> vijaypkuma...@gmail.com> wrote: >> >> Dear all, >> >> I am trying to crystallize a 30 kD protein. Protein crystals are formed >> after 3 months. The crystals are formed in the following condition: >> 0.01M Zinc sulphate >> 0.1M MES monohydrate pH 6.5 >> 25% v/v PEG monomethyl ether 550 >> >> Please suggest me how to grow these crystals faster. >> >> Thanking you >> >> -- >> Vijaykumar Pillalamarri, >> UGC-JRF, >> C/O: Dr. Anthony Addlagatta, >> Senior Scientist, >> CSIR-IICT, Tarnaka, >> Hyderabad, India-57 >> Mobile: +918886922975 >> >> >> > > > -- > patr...@douglas.co.ukDouglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090US toll
Re: [ccp4bb] fastening crystal formation
Thank you all for your replies. I tried seeding also. But it did not help me. I ran SDS-PAGE. The fresh protein and crystal are showing bands at same molecular weight. So there might not be protein degradation. On 3 November 2014 22:24, Appu kumar wrote: > Hello, > If the homologous protein structure is available, or you have symmetry > information of your crystal, then try engineering surface by putting in > residues which has less entropy like replacing Lys to Arg, Met to Gln. We > are able to fasten the crystal growth from 20 days to 3-4 days by creating > phenylalanine Pi-stack. This is tricky but if you have an idea of crystal > contact, then it should be straightforward without much trouble. > Good Luck > Appu > > On 31 October 2014 11:43, Patrick Shaw Stewart > wrote: > >> >> Michael (and some others) >> >> You haven't mentioned - and I guess don't use regularly - the "random" >> MMS approach, where crushed seed-crystals are added to random screens. >> This really is a terrific method, and it will on average roughly double >> productivity. It's the first thing I would think of in a case like >> Vijaykumar's (as I told him this morning!). >> >> Galina Obmolova and her colleagues published a great paper earlier this >> year about MMS. They were working with a set of 16 Fab fragments that >> comprised all combinations of 4 different heavy chains and 4 different >> light chains. Three structures were generated without MMS, but by various >> very creative uses of microseeding they were able to get all 16/16 >> structures. Ref below. >> >> Best wishes, >> >> Patrick >> >> __ >> >> >> Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., & Gilliland, G. >> L. (2014). Protein crystallization with microseed matrix screening: >> application to human germline antibody Fabs. *Structural Biology and >> Crystallization Communications*, *70*(8). >> >> >> >> >> On 31 October 2014 16:07, R. M. Garavito wrote: >> >>> Although three months is a long time, it is no completely unheard of, >>> and does not require the invocation of proteolysis. The longest time I >>> have heard of is ~1 yr, so count yourself lucky. However to get good >>> advice, as well as to use it, you need to ask yourself several questions: >>> >>> 1. What kind of crystals are they? Protein, salt, etc? If they are >>> salt, don't pursue this condition. >>> >>> 2. How many crystals did you get? One or 2 in a drop or a microcrystal >>> shower. And of what kind? Single, well shaped, rosettes, needle clusters, >>> or something that looks crystalline. Screen more broadly around your >>> initial hit. >>> >>> 3. How many times have your tried to repeat this? Once, twice, or >>> more? Did you try setups in duplicate? If so, did you get reproducible >>> results? Have you actually screened around these conditions, varying each >>> component systematically (PEG, salt, pH, buffer, etc.)? >>> >>> 4. What method did you use? And in what kind of container? For one >>> thing, we don't completely trust the integrity of our setups for longer >>> than 2 months. All containers leak water slowly, so when crystals take >>> longer than 2 months to grow (a) the real conditions are at much higher >>> values than you naively think (i.e., the drop has dried out more than you >>> expected) or (b) other components are crystallizing, for example a zinc >>> salt. It depends what else is in your protein buffer, as well. >>> >>> To quicken protein crystallization (which is not always a good thing), >>> increase your protein concentration (by 1.5-2x) and/or PEG concentration >>> (such as screening up to 40% PEGmme 550). Sadly, crystallization is a >>> combination of thermodynamic and kinetic factors: you can get crystals >>> (sometimes a single crystal only) when just outside the truly optimal >>> conditions, but this may be only a sporadic event. You got to keep >>> screening. >>> >>> Good luck, >>> >>> Michael >>> >>> >>> ** >>> *R. Michael Garavito, Ph.D.* >>> *Professor of Biochemistry & Molecular Biology* >>> *603 Wilson Rd., Rm. 513* >>> *Michigan State University * >>> *East Lansing, MI 48824-1319* >>> *Office:* *(517) 355-9724 <%28517%29%20355-9724> Lab: (517) >>> 353-9125 <%28517%29%20353-9125>* >>> *FAX: (517) 353-9334 <%28517%29%20353-9334> >>> Email: rmgarav...@gmail.com * >>> ** >>> >>> >>> >>> >>> On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < >>> vijaypkuma...@gmail.com> wrote: >>> >>> Dear all, >>> >>> I am trying to crystallize a 30 kD protein. Protein crystals are formed >>> after 3 months. The crystals are formed in the following condition: >>> 0.01M Zinc sulphate >>> 0.1M MES monohydrate pH 6.5 >>> 25% v/v PEG monomethyl ether 550 >>> >>> Please suggest me how to grow these crystals faster. >>> >>> Thanking you >>> >>> -- >>> Vijaykumar Pillalamarri, >>> UGC-JR
