Re: [ccp4bb] Flexible C terminus
Hi Chitra, Sometimes disorder ‘is’ the functional state of a peptide segment, and in those cases, if you see the segment ordered in your crystal structure, it is not physiologically relevant; rather it is a result of crystal packing. Sometimes disordered segments undergo a folding when they bind a ligand, or they are targets for post-translational modifications, and in some cases they can actually generate an entropic force which can modify the structural ensemble of the protein (https://www.nature.com/articles/s41586-018-0699-5). In the latter case, the protein had a disordered 30 residue C-terminus that used entropic force to change the structure of the folded portion of the protein to enhance ligand affinity. Best regards, Z *** Zachary A. Wood, Ph.D. Associate Professor and Graduate Coordinator Department of Biochemistry & Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab:706-583-0303 FAX: 706-542-1738 *** From: CCP4 bulletin board on behalf of Debanu Das Reply-To: "debanu@gmail.com" Date: Thursday, March 12, 2020 at 12:53 PM To: "CCP4BB@JISCMAIL.AC.UK" Subject: Re: [ccp4bb] Flexible C terminus [EXTERNAL SENDER - PROCEED CAUTIOUSLY] Hi Chitra, To add to the discussion, I can offer an example about obtaining the structure of a flexible/disordered N-term of a membrane protein. Previous structures of the full-length multidrug efflux transporter AcrB (12 TM helices in each protein ~1000 residues, forms a trimer, so 36 TM helices) were missing the first 6 residues in the N-term in the cytoplasm but we could determine this structure and look at some interesting sequence-structure implications of these first 6 residues in our structure: "Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å resolution reveals the N-terminal region with conserved amino acids Debanu Das,* Qian Steven Xu,* Jonas Y. Lee, Irina Ankoudinova, Candice Huang, Yun Lou, Andy DeGiovanni, Rosalind Kim, and Sung-Hou Kim" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2023878/ I think there should also be examples of C-term or N-term expression/purification tags that are ordered in some crystal forms of a target but disordered in other crystal forms/structures of the same target/homologs. Best, Debanu -- Debanu Das On Thu, Mar 12, 2020 at 5:02 AM chitra latka mailto:chitra.la...@gmail.com>> wrote: Dear Klemens, I am going to setup the crystallisation of the entire protein anyhow. I hope I get lucky :) Thanks Chitra On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild mailto:klemens.w...@bzh.uni-heidelberg.de>> wrote: On 12.03.20 08:53, chitra latka wrote: Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 Dear Chitra I would nevertheless try. Sometimes flexible termini fold back either in cis or in trans (crystal packing, a case I just had Yesterday) and you might learn sth important for biological regulation if you are lucky. At the same time I would truncate the terminus and crystallize the globular domain in parallel. Good luck Klemens -- Regards Chitra To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Flexible C terminus
Hi Chitra, To add to the discussion, I can offer an example about obtaining the structure of a flexible/disordered N-term of a membrane protein. Previous structures of the full-length multidrug efflux transporter AcrB (12 TM helices in each protein ~1000 residues, forms a trimer, so 36 TM helices) were missing the first 6 residues in the N-term in the cytoplasm but we could determine this structure and look at some interesting sequence-structure implications of these first 6 residues in our structure: "Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å resolution reveals the N-terminal region with conserved amino acids Debanu Das,* Qian Steven Xu,* Jonas Y. Lee, Irina Ankoudinova, Candice Huang, Yun Lou, Andy DeGiovanni, Rosalind Kim, and Sung-Hou Kim" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2023878/ I think there should also be examples of C-term or N-term expression/purification tags that are ordered in some crystal forms of a target but disordered in other crystal forms/structures of the same target/homologs. Best, Debanu -- Debanu Das On Thu, Mar 12, 2020 at 5:02 AM chitra latka wrote: > Dear Klemens, > > I am going to setup the crystallisation of the entire protein anyhow. I > hope I get lucky :) > > Thanks > Chitra > > On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild < > klemens.w...@bzh.uni-heidelberg.de> wrote: > >> On 12.03.20 08:53, chitra latka wrote: >> >> Dear All, >> >> I am working on a protein that has flexible C terminus. None of the >> available structures even in homologs have density for C term region >> (around 20 odd residues). All the available pdb entries have missing >> density for these 20 residues at C terminus. >> >> I am going to try my luck crystallising the entire protein in hope of >> getting density for C term residues as well (Fingers crossed). >> >> Has anyone faced a similar problem where they have managed to get density >> for a flexible terminus successfully? >> >> Any suggestions would be appreciated. >> >> Cheers ! >> >> Chitra Latka >> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> >> Dear Chitra >> >> I would nevertheless try. Sometimes flexible termini fold back either in >> cis or in trans (crystal packing, a case I just had Yesterday) and you >> might learn sth important for biological regulation if you are lucky. At >> the same time I would truncate the terminus and crystallize the globular >> domain in parallel. >> >> Good luck >> >> Klemens >> > > > -- > Regards > Chitra > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Flexible C terminus
Dear Chitra, Try adding a ligand to the crystallization. This worked for us with ALDH7A1: https://www.ncbi.nlm.nih.gov/pubmed/26260980 ALDH7A1 is an example of an enzyme with a flexible C-terminus (11 residues). The conformation of the C-terminus depends on the presence of a ligand in the substrate site. With the product bound, the C-terminus adopts the “in” conformation (4ZUL). Without the product bound, the C-terminus adopts either the “out” conformation (4ZUK, chains A-F,H) or is disordered (4ZUK, chain G). In this case, the flexibility of the C-terminus is essential for catalytic activity:. https://www.ncbi.nlm.nih.gov/pubmed/29045138 Good luck! Jack John J. Tanner Professor of Biochemistry and Chemistry Associate Chair of Biochemistry University of Missouri 117 Schweitzer Hall 503 S. College Ave. Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-5635 Email: tanne...@missouri.edu<mailto:tanne...@missouri.edu> http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html Lab: Schlundt Annex rooms 3,6,9, 203B, 203C Office: Schlundt Annex 203A On Mar 12, 2020, at 8:01 AM, Oganesyan, Vaheh mailto:vaheh.oganes...@astrazeneca.com>> wrote: Hi Chitra Latka, By far the best approach is to find what protein is interacting with the one you have the structure and try co-crystallizing them together. At least there will be some more biology (science) involved in what you will be doing. You may get lucky and get different packing of your full length protein and get some sort of structure for last 20 aa. Then what? Regards, From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> On Behalf Of chitra latka Sent: Thursday, March 12, 2020 3:54 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Flexible C terminus Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://clicktime.symantec.com/3K2tGPDPQHVzmmXDiGLP6PM6H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Flexible C terminus
Hi Chitra Latka, By far the best approach is to find what protein is interacting with the one you have the structure and try co-crystallizing them together. At least there will be some more biology (science) involved in what you will be doing. You may get lucky and get different packing of your full length protein and get some sort of structure for last 20 aa. Then what? Regards, From: CCP4 bulletin board On Behalf Of chitra latka Sent: Thursday, March 12, 2020 3:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Flexible C terminus Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://clicktime.symantec.com/3K2tGPDPQHVzmmXDiGLP6PM6H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus
Dear Chitra, Crystallizing both proteins together is a very good idea: If you get a structure, it will be an interesting one, even if the C-terminus remains invisible. Concerning the flexible C-terminus: is it the linker linking the second domain to the first one? In that case it might just be a bait, attracting a protease to get the second domain chopped off. In that case, chances of getting it structured may be slim and if you get it structured, it might be a crystal packing artifact. However, as Klemens mentioned, you should nevertheless try! If this flexible C-terminus gets cleaved by a protease, you may also try to get some cocrystal structure with the protease. There are quite a few tricks available to achieve this. Best, Herman Von: chitra latka Gesendet: Donnerstag, 12. März 2020 12:59 An: Schreuder, Herman /DE Cc: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [EXTERNAL] [ccp4bb] Flexible C terminus EXTERNAL : Real sender is chitra.la...@gmail.com<mailto:chitra.la...@gmail.com> Dear Herman, Its a two domain protein and the second domain gets chopped off and stabilises the other domain by binding near the flexible C - terminus of first domain. I am trying to crystallise both the proteins together. Literature review doesn't report binding of the protein to any other protein. So, it kind of has the ligand or another domain to stabilise but eve then density of C-terminus residues remain missing. Thanks Chitra Latka On Thu, Mar 12, 2020 at 4:08 PM Schreuder, Herman /DE mailto:herman.schreu...@sanofi.com>> wrote: Dear Chitra, There usually is a reason the C-terminus is disordered. Either it needs to bind a ligand to get ordered, or it needs to bind to some other protein. You have to check the literature. If the C-terminus binds a ligand, you have to add this ligand to your crystallization experiment. If it binds to some other protein, you could try cocrystallization of both proteins, or just try to cocrystallize the other protein with only the 20 residues or so of the C-terminus. Best, Herman Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> Im Auftrag von chitra latka Gesendet: Donnerstag, 12. März 2020 08:54 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk> Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=tHXRPIRekvSgMSDPEVP15ta2iH1g8NkqcZVMEpk1VLo=k0Yuiu6SCu5YkIBlnejKmh7Eq9F_cX3TLtdqQZVyR3w=> -- Regards Chitra To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Flexible C terminus
Dear Klemens, I am going to setup the crystallisation of the entire protein anyhow. I hope I get lucky :) Thanks Chitra On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild < klemens.w...@bzh.uni-heidelberg.de> wrote: > On 12.03.20 08:53, chitra latka wrote: > > Dear All, > > I am working on a protein that has flexible C terminus. None of the > available structures even in homologs have density for C term region > (around 20 odd residues). All the available pdb entries have missing > density for these 20 residues at C terminus. > > I am going to try my luck crystallising the entire protein in hope of > getting density for C term residues as well (Fingers crossed). > > Has anyone faced a similar problem where they have managed to get density > for a flexible terminus successfully? > > Any suggestions would be appreciated. > > Cheers ! > > Chitra Latka > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > Dear Chitra > > I would nevertheless try. Sometimes flexible termini fold back either in > cis or in trans (crystal packing, a case I just had Yesterday) and you > might learn sth important for biological regulation if you are lucky. At > the same time I would truncate the terminus and crystallize the globular > domain in parallel. > > Good luck > > Klemens > -- Regards Chitra To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] [EXTERNAL] [ccp4bb] Flexible C terminus
Dear Herman, Its a two domain protein and the second domain gets chopped off and stabilises the other domain by binding near the flexible C - terminus of first domain. I am trying to crystallise both the proteins together. Literature review doesn't report binding of the protein to any other protein. So, it kind of has the ligand or another domain to stabilise but eve then density of C-terminus residues remain missing. Thanks Chitra Latka On Thu, Mar 12, 2020 at 4:08 PM Schreuder, Herman /DE < herman.schreu...@sanofi.com> wrote: > Dear Chitra, > > > > There usually is a reason the C-terminus is disordered. Either it needs to > bind a ligand to get ordered, or it needs to bind to some other protein. > You have to check the literature. If the C-terminus binds a ligand, you > have to add this ligand to your crystallization experiment. If it binds to > some other protein, you could try cocrystallization of both proteins, or > just try to cocrystallize the other protein with only the 20 residues or so > of the C-terminus. > > > > Best, Herman > > > > *Von:* CCP4 bulletin board *Im Auftrag von *chitra > latka > *Gesendet:* Donnerstag, 12. März 2020 08:54 > *An:* CCP4BB@JISCMAIL.AC.UK > *Betreff:* [EXTERNAL] [ccp4bb] Flexible C terminus > > > > *EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk > > > > Dear All, > > > > I am working on a protein that has flexible C terminus. None of the > available structures even in homologs have density for C term region > (around 20 odd residues). All the available pdb entries have missing > density for these 20 residues at C terminus. > > > > I am going to try my luck crystallising the entire protein in hope of > getting density for C term residues as well (Fingers crossed). > > > > Has anyone faced a similar problem where they have managed to get density > for a flexible terminus successfully? > > > > Any suggestions would be appreciated. > > > > Cheers ! > > > > Chitra Latka > > > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=tHXRPIRekvSgMSDPEVP15ta2iH1g8NkqcZVMEpk1VLo=k0Yuiu6SCu5YkIBlnejKmh7Eq9F_cX3TLtdqQZVyR3w=> > -- Regards Chitra To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Flexible C terminus
On 12.03.20 08:53, chitra latka wrote: Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 Dear Chitra I would nevertheless try. Sometimes flexible termini fold back either in cis or in trans (crystal packing, a case I just had Yesterday) and you might learn sth important for biological regulation if you are lucky. At the same time I would truncate the terminus and crystallize the globular domain in parallel. Good luck Klemens To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus
Dear Chitra, There usually is a reason the C-terminus is disordered. Either it needs to bind a ligand to get ordered, or it needs to bind to some other protein. You have to check the literature. If the C-terminus binds a ligand, you have to add this ligand to your crystallization experiment. If it binds to some other protein, you could try cocrystallization of both proteins, or just try to cocrystallize the other protein with only the 20 residues or so of the C-terminus. Best, Herman Von: CCP4 bulletin board Im Auftrag von chitra latka Gesendet: Donnerstag, 12. März 2020 08:54 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk<mailto:owner-ccp...@jiscmail.ac.uk> Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=tHXRPIRekvSgMSDPEVP15ta2iH1g8NkqcZVMEpk1VLo=k0Yuiu6SCu5YkIBlnejKmh7Eq9F_cX3TLtdqQZVyR3w=> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Flexible C terminus
Dear All, I am working on a protein that has flexible C terminus. None of the available structures even in homologs have density for C term region (around 20 odd residues). All the available pdb entries have missing density for these 20 residues at C terminus. I am going to try my luck crystallising the entire protein in hope of getting density for C term residues as well (Fingers crossed). Has anyone faced a similar problem where they have managed to get density for a flexible terminus successfully? Any suggestions would be appreciated. Cheers ! Chitra Latka To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
