[ccp4bb] protein degradation in crystal
Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa
Re: [ccp4bb] protein degradation in crystal
Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth wrote: > Dear Lisa > It is not uncommon to see breakdown products when you run crystals on > gel. Espesially if they are older crystals, sometimes you even see higher > molecular bands, these are probably due to intra molecular cross links > formed over time. > If you are worried about stability, try to increase the crystallization > speed, we have one example where we see a clear difference in both crystal > quality and even space group depending on when we fish the crystals. The > crystals appear within 5 min, the best quality data sets come from > crystals we fish after only 30-60 min. > You may also have a little protease contamination of course, to prevent > this add protease inhibitor, or DTT, or EDTA to you protein before you set > it up. > cheers Preben > > > On 1/16/13 12:14 PM, LISA wrote: > >> Hi All, >> I have an 36KD protein which can be crystallize in two days. Most of the >> crystals are very big. But all cystals have poor resolution,lower than 3.8 >> A. I picked some crystals, washed them in the mother solution and then run >> SDS-PAGE. It is surprised to find that different cystals have different >> components. Some crystals have several samll bands below the band of the >> protein. And in some crysals the bigger size band (as the construct should >> be) almost disappared and have smear. Does the protein was degradated in >> the crystals? Did someone met the similar problem as I? Thanks >> >> All the best >> lisa >> > > -- > J. Preben Morth, Ph.D > Group Leader > Membrane Transport Group > Nordic EMBL Partnership > Centre for Molecular Medicine Norway (NCMM) > University of Oslo > P.O.Box 1137 Blindern > 0318 Oslo, Norway > > Email: j.p.mo...@ncmm.uio.no > Tel: +47 2284 0794 > > http://www.jpmorth.dk >
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of John Domsic Sent: Wednesday, January 16, 2013 2:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth wrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex. Tom ** > Hi John, > > This is really an amazing wild west story: the man who crystallizes faster > than his protease! I really must compliment you with how you successfully > performed these experiments! > > Unfortunately, proteins usually do not crystallize that fast (at least not > in my hands), so in these cases other methods have to be used. As has > mentioned before, protease inhibitors are the way to go. Especially with > autolysis, as one protease cuts another one, the speed of the reaction goes > with the square of the protease concentration. Whereas in dilute solutions > not much happens, as soon as you start to concentrate towards > crystallization conditions, say 10 mg/ml, degradation suddenly goes very > fast. > > There are 2 cases to consider: > 1) the protein you want to crystallize is a protease and is destroying > itself. In this case you need to cocrystallize with a potent and specific > inhibitor. With serine proteases, Wolfram Bode was very successful by using > chloromethylketone-containing peptides (e.g. PPACK). These compounds would > make covalent links with both the active site serine and histidine, > effectively killing any protease activity. > > 2) the protein you want to crystallize is not a protease and it is a > contaminant which is causing the problems. In this case I would add a > protease inhibitor coctail in an earlier step of the purification to block > the protease before the final purification steps. I would also add some > small broad protease inhibitor e.g. PMSF to the protein solution used for > crystallization. > > Herman > > -- > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John > Domsic > *Sent:* Wednesday, January 16, 2013 2:22 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] protein degradation in crystal > > Hi Lisa, > > Speed is definitely a big factor here. With a protein I work with I can > get large crystals in myriad conditions that only diffract to about 4-5 > Ang. What I ended up doing was taking these crystals and seeding entire > screens. I found that not only would crystals appear sooner but it > revealed novel crystallization conditions. These seeded crystals would > appear within minutes as Preben described and diffracted to better than 2 > Ang. Another thought would be to try limited proteolysis to see if you can > identify a more stable construct. > > -John > > -- > > John Domsic > Postdoctoral Fellow > Gene Expression and Regulation Program > The Wistar Institute > Philadelphia, PA 19104 > > > On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth > wrote: > >> Dear Lisa >> It is not uncommon to see breakdown products when you run crystals on >> gel. Espesially if they are older crystals, sometimes you even see higher >> molecular bands, these are probably due to intra molecular cross links >> formed over time. >> If you are worried about stability, try to increase the crystallization >> speed, we have one example where we see a clear difference in both crystal >> quality and even space group depending on when we fish the crystals. The >> crystals appear within 5 min, the best quality data sets come from >> crystals we fish after only 30-60 min. >> You may also have a little protease contamination of course, to prevent >> this add protease inhibitor, or DTT, or EDTA to you protein before you set >> it up. >> cheers Preben >> >> >> On 1/16/13 12:14 PM, LISA wrote: >> >>> Hi All, >>> I have an 36KD protein which can be crystallize in two days. Most of the >>> crystals are very big. But all cystals have poor resolution,lower than 3.8 >>> A. I picked some crystals, washed them in the mother solution and then run >>> SDS-PAGE. It is surprised to find that different cystals have different >>> components. Some crystals have several samll bands below the band of the >>> protein. And in some crysals the bigger size band (as the construct should >>> be) almost disappared and have smear. Does the protein was degradated in >>> the crystals? Did someone met the similar problem as I? Thanks >>> >>> All the best >>> lisa >>> >> >> -- >> J. Preben Morth, Ph.D >> Group Leader >> Membrane Transport Group >> Nordic EMBL Partnership >> Centre for Molecular Medicine Norway (NCMM) >> University of Oslo >> P.O.Box 1137 Blindern >> 0318 Oslo, Norway >> >> Email: j.p.mo...@ncmm.uio.no >> Tel: +47 2284 0794 <%2B47%202284%200794> >> >> http://www.jpmorth.dk >> > > -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
Re: [ccp4bb] protein degradation in crystal
Could you identify the cleavage sites by protein sequencing and design new constructs (truncated versions) accordingly? It might improve your crystal quality to get better resolution. Joe On Wed, Jan 16, 2013 at 9:22 AM, Tom Murray-Rust wrote: > Just to add to Herman's suggestions, if you are trying to crystallise a > protease then you could also try using the S195A variant rather than an > inhibitor. This would certainly be the case if you ever want to > co-crystallise in a substrate, as PPACK (or the like) would occupy the > active site cleft and prevent formation of the protease-substrate complex. > > Tom > > > > ** >> Hi John, >> >> This is really an amazing wild west story: the man who crystallizes >> faster than his protease! I really must compliment you with how you >> successfully performed these experiments! >> >> Unfortunately, proteins usually do not crystallize that fast (at least >> not in my hands), so in these cases other methods have to be used. As has >> mentioned before, protease inhibitors are the way to go. Especially with >> autolysis, as one protease cuts another one, the speed of the reaction goes >> with the square of the protease concentration. Whereas in dilute solutions >> not much happens, as soon as you start to concentrate towards >> crystallization conditions, say 10 mg/ml, degradation suddenly goes very >> fast. >> >> There are 2 cases to consider: >> 1) the protein you want to crystallize is a protease and is destroying >> itself. In this case you need to cocrystallize with a potent and specific >> inhibitor. With serine proteases, Wolfram Bode was very successful by using >> chloromethylketone-containing peptides (e.g. PPACK). These compounds would >> make covalent links with both the active site serine and histidine, >> effectively killing any protease activity. >> >> 2) the protein you want to crystallize is not a protease and it is a >> contaminant which is causing the problems. In this case I would add a >> protease inhibitor coctail in an earlier step of the purification to block >> the protease before the final purification steps. I would also add some >> small broad protease inhibitor e.g. PMSF to the protein solution used for >> crystallization. >> >> Herman >> >> ---------- >> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of >> *John Domsic >> *Sent:* Wednesday, January 16, 2013 2:22 PM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* Re: [ccp4bb] protein degradation in crystal >> >> Hi Lisa, >> >> Speed is definitely a big factor here. With a protein I work with I can >> get large crystals in myriad conditions that only diffract to about 4-5 >> Ang. What I ended up doing was taking these crystals and seeding entire >> screens. I found that not only would crystals appear sooner but it >> revealed novel crystallization conditions. These seeded crystals would >> appear within minutes as Preben described and diffracted to better than 2 >> Ang. Another thought would be to try limited proteolysis to see if you can >> identify a more stable construct. >> >> -John >> >> -- >> >> John Domsic >> Postdoctoral Fellow >> Gene Expression and Regulation Program >> The Wistar Institute >> Philadelphia, PA 19104 >> >> >> On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth > > wrote: >> >>> Dear Lisa >>> It is not uncommon to see breakdown products when you run crystals on >>> gel. Espesially if they are older crystals, sometimes you even see higher >>> molecular bands, these are probably due to intra molecular cross links >>> formed over time. >>> If you are worried about stability, try to increase the crystallization >>> speed, we have one example where we see a clear difference in both crystal >>> quality and even space group depending on when we fish the crystals. The >>> crystals appear within 5 min, the best quality data sets come from >>> crystals we fish after only 30-60 min. >>> You may also have a little protease contamination of course, to prevent >>> this add protease inhibitor, or DTT, or EDTA to you protein before you set >>> it up. >>> cheers Preben >>> >>> >>> On 1/16/13 12:14 PM, LISA wrote: >>> >>>> Hi All, >>>> I have an 36KD protein which can be crystallize in two days. Most of >>>> the crystals are very big. But all cystals have poor resolution,lower than >>>> 3.8 A. I picked some cr
