Re: [ccp4bb] changes in small sections of secondary structure
Being of an untrusting disposition, I would ask your collaborator for the reflection data as well as the PDBs - it is always a good idea to look at the maps when there is some unexpected structural feature! - The DB provides an electron density server, if the structures have been deposited, otherwise it is straightforward to generate a map yourself and look at it in coot. Eleanor Dodson On 21 October 2013 13:53, Antony Oliver wrote: > Dear Mahesh, > > Are all the structures at similar resolution? Definition of secondary > structure is, and can be, affected by the level of geometric > restraints/constraints used in the refinement process. > > Tony. > > --- > Dr Antony W Oliver > Senior Research Fellow > CR-UK DNA Repair Enzymes Group > Genome Damage and Stability Centre > Science Park Road > University of Sussex > Falmer, Brighton, BN1 9RQ > > email: antony.oli...@sussex.ac.uk > tel (office): +44 (0)1273 678349 > tel (lab): +44 (0)1273 677512 > > On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote: > >> Hello experts >> >> Thanks for your insights. >> For one of the structures, it turned out to be a rendering issue by pymol >> like matt pointed out. For the other, the residues are clearly in a less >> than ideal position. Even if I see deviation from the RMSD plots, i cannot >> be sure that the structure were refined ideally at those positions ( those >> are not my structures, i just have the pdb files from my collaborator). >> >> Thanks again, >> >> Mahesh >> >> P.S from what all of you are saying it sounds like those changes are not >> real, if I find that they could be Ill let everyone know. >> >> >> >> >>
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, Are all the structures at similar resolution? Definition of secondary structure is, and can be, affected by the level of geometric restraints/constraints used in the refinement process. Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote: > Hello experts > > Thanks for your insights. > For one of the structures, it turned out to be a rendering issue by pymol > like matt pointed out. For the other, the residues are clearly in a less than > ideal position. Even if I see deviation from the RMSD plots, i cannot be sure > that the structure were refined ideally at those positions ( those are not my > structures, i just have the pdb files from my collaborator). > > Thanks again, > > Mahesh > > P.S from what all of you are saying it sounds like those changes are not > real, if I find that they could be Ill let everyone know. > > > > >
Re: [ccp4bb] changes in small sections of secondary structure
Hello experts Thanks for your insights. For one of the structures, it turned out to be a rendering issue by pymol like matt pointed out. For the other, the residues are clearly in a less than ideal position. Even if I see deviation from the RMSD plots, i cannot be sure that the structure were refined ideally at those positions ( those are not my structures, i just have the pdb files from my collaborator). Thanks again, Mahesh P.S from what all of you are saying it sounds like those changes are not real, if I find that they could be Ill let everyone know. >
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, You could also run the CCP4 program CONTACT/ACT (Tadeusz Skarzynski & Andrew Leslie/Wojtek Rypniewski & Howard Terry) for inter residue contacts and compare the NH-OC bonds in the different structures. Best, D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Swastik Phulera Sent: 21 October 2013 07:52 To: ccp4bb Subject: Re: [ccp4bb] changes in small sections of secondary structure Dear Mahesh, Instead of bothering with the secondary structure assignments why may try to superpose your structures and then view your RMSD plots(Superpose program in CCP4 can do the trick). You might then be better off focusing on the regions with high RMSD. Regards On Sat, Oct 19, 2013 at 3:20 AM, krish wrote: > Hi Mahesh, > > If you have made these screen shots using pymol, then you really need > to be careful before you jump to conclude anything from these > secondary structural changes (Matt already mentioned it). Assign > secondary structures using DSSP or see the secondary structure restraints in > the structure while refining. > Manual refining of these areas is really important (I mean careful > inspection) and once you think these regions you highlighted have > proper restraints, then you can conclude to a CERTAIN extent. > > I would perform MD simulation runs after energy minimization for each > structure for 10 to 15 nanoseconds and see whether these small > perturbations (what you have depicted) are really there. These runs > with identical parameters should give you clear hints how much your > protein in different states is stable with time. Also, you can figure > out other structural changes with longer simulation times if at all if your > protein is dynamic. > > Hope this helps! > > Krish > > > On Fri, Oct 18, 2013 at 2:52 PM, Mahesh Lingaraju wrote: >> >> Hello experts >> >> Attached is an image showing different crystal structures of the same >> protein in diffrent states (mutant vs substrate bound vs unliganded) >> and highlighted are little changes in secondary structure. I was >> wondering how real such small changes are and if they are real could >> they be enough to perturb the energy potential of the protein significantly. >> I apologize if this is a naive question as this is clearly not my >> area of expertise. >> >> Thanks for your input >> >> Mahesh >> >> >> >> > > -- -- स्वस्तिक फुलेरा वरिष्ठ अनुसंधान कर्ता संरचनात्मक जीवविज्ञान प्रयोगशाला डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र हैदराबाद, ५००, ००१ और राष्ट्रीय कोशिका विज्ञान केंद्र पुणे विश्वविद्यालय परिसर, गणेशखिंड पुना, ४११,००७
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, In addition to RMSD plots, you could try to quantitatively analyse the protein residue flexibility in order to detect backbone conformational transitions by means of the method proposed in: Local Fluctuations and Conformational Transitions in Proteins Rocco Caliandro, Giulia Rossetti, and Paolo Carloni J. Chem. Theory Comput. 2012, 8, 4775−4785 By starting from a set of several models of the same protein, the method allows to detect the "hinge points" of the protein and the residues where transitions between two different conformational states occur. However, by inspecting your picture, also I am not convinced that they are alterations of the protein, but rather inconsistency in assignment of secondary structure. Danilo
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, Instead of bothering with the secondary structure assignments why may try to superpose your structures and then view your RMSD plots(Superpose program in CCP4 can do the trick). You might then be better off focusing on the regions with high RMSD. Regards On Sat, Oct 19, 2013 at 3:20 AM, krish wrote: > Hi Mahesh, > > If you have made these screen shots using pymol, then you really need to be > careful before you jump to conclude anything from these secondary structural > changes (Matt already mentioned it). Assign secondary structures using DSSP > or see the secondary structure restraints in the structure while refining. > Manual refining of these areas is really important (I mean careful > inspection) and once you think these regions you highlighted have proper > restraints, then you can conclude to a CERTAIN extent. > > I would perform MD simulation runs after energy minimization for each > structure for 10 to 15 nanoseconds and see whether these small perturbations > (what you have depicted) are really there. These runs with identical > parameters should give you clear hints how much your protein in different > states is stable with time. Also, you can figure out other structural > changes with longer simulation times if at all if your protein is dynamic. > > Hope this helps! > > Krish > > > On Fri, Oct 18, 2013 at 2:52 PM, Mahesh Lingaraju wrote: >> >> Hello experts >> >> Attached is an image showing different crystal structures of the same >> protein in diffrent states (mutant vs substrate bound vs unliganded) and >> highlighted are little changes in secondary structure. I was wondering how >> real such small changes are and if they are real could they be enough to >> perturb the energy potential of the protein significantly. >> I apologize if this is a naive question as this is clearly not my area of >> expertise. >> >> Thanks for your input >> >> Mahesh >> >> >> >> > > -- -- स्वस्तिक फुलेरा वरिष्ठ अनुसंधान कर्ता संरचनात्मक जीवविज्ञान प्रयोगशाला डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र हैदराबाद, ५००, ००१ और राष्ट्रीय कोशिका विज्ञान केंद्र पुणे विश्वविद्यालय परिसर, गणेशखिंड पुना, ४११,००७
Re: [ccp4bb] changes in small sections of secondary structure
Hi Mahesh, If you have made these screen shots using pymol, then you really need to be careful before you jump to conclude anything from these secondary structural changes (Matt already mentioned it). Assign secondary structures using DSSP or see the secondary structure restraints in the structure while refining. Manual refining of these areas is really important (I mean careful inspection) and once you think these regions you highlighted have proper restraints, then you can conclude to a CERTAIN extent. I would perform MD simulation runs after energy minimization for each structure for 10 to 15 nanoseconds and see whether these small perturbations (what you have depicted) are really there. These runs with identical parameters should give you clear hints how much your protein in different states is stable with time. Also, you can figure out other structural changes with longer simulation times if at all if your protein is dynamic. Hope this helps! Krish On Fri, Oct 18, 2013 at 2:52 PM, Mahesh Lingaraju wrote: > Hello experts > > Attached is an image showing different crystal structures of the same > protein in diffrent states (mutant vs substrate bound vs unliganded) and > highlighted are little changes in secondary structure. I was wondering how > real such small changes are and if they are real could they be enough to > perturb the energy potential of the protein significantly. > I apologize if this is a naive question as this is clearly not my area of > expertise. > > Thanks for your input > > Mahesh > > > > >
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh - I can't comment on the energy changes for the protein, but I can say that the changes you've highlighted are not really due to alterations of the protein, but rather inconsistency in assignment of secondary structure. If you look closely at the areas you've highlighted, you will see that the C-alpha backbone as depicted in this cartoon rendering is still making a helix even in the structures where it is not drawn as an alpha-helix. This typically means that some residues have just fallen below whatever threshold the rendering program uses to define secondary structure (like position on a Ramachandran plot) in one structure, and the same residues are just above the threshold in the other structure. This is not a significant change. Indeed, different programs may render the same structure differently. I hope that clears up some confusion. All the best, Matt On 10/18/13 2:52 PM, Mahesh Lingaraju wrote: Hello experts Attached is an image showing different crystal structures of the same protein in diffrent states (mutant vs substrate bound vs unliganded) and highlighted are little changes in secondary structure. I was wondering how real such small changes are and if they are real could they be enough to perturb the energy potential of the protein significantly. I apologize if this is a naive question as this is clearly not my area of expertise. Thanks for your input Mahesh -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
