Re: [ccp4bb] Resolution discrepancy between MTZ file and Refinement
Hi Thank you Pavel for your input. Venkat On Wed, Apr 10, 2024 at 1:44 AM Pavel Afonine wrote: > Hi, > every time you run phenix.refine it may exclude some reflections from > refinement per Read 1999 (Acta Cryst. (1999). D55, 1759-1764). Usually the > number of reflections omitted ranges from none to just a few, however, this > may be just enough to make the resolution statistics look slightly > different. > Also, if you use Iobs as input data, internally they are converted into > Fobs using F method, and some reflections may not "survive" this > conversion. This may be another reason for discrepancies. > Pavel > > On Fri, Apr 5, 2024 at 9:45 PM venkatareddy dadireddy < > venkatda...@gmail.com> wrote: > >> >> Thank you very much Garib. >> >> Venkat >> >> On Sat, Apr 6, 2024 at 1:12 AM Garib Murshudov >> wrote: >> >>> Unless you are confident that twin exists you should not use twin >>> refinement (Occam’s razor) >>> >>> >>> >>> On 5 Apr 2024, at 17:24, venkatareddy dadireddy >>> wrote: >>> >>> CAUTION: This email originated from outside of the LMB: >>> *.-owner-ccp...@jiscmail.ac.uk -.* >>> Do not click links or open attachments unless you recognize the sender >>> and know the content is safe. >>> If you think this is a phishing email, please forward it to >>> phish...@mrc-lmb.cam.ac.uk >>> >>> >>> -- >>> Hi Kay and Garib, >>> >>> Thank you for your input. >>> It is actually the twin refinement that gave rise to resolution >>> discrepancy. >>> For what reason I don't remember that I have turned the twin refinement >>> ON >>> and the same job was cloned again and again. >>> With the twin refinement OFF, it gave rise to resolution present in the >>> MTZ (2.0 A). >>> From Xtriage: *the correlation between* >>> *the intensities related by the twin law 1/2*h-3/2*k, -1/2*h-1/2*k,-l >>> with an estimated twin* >>> *fraction of 0.10 is most likely due to an NCS axis parallel to the twin >>> axis*. >>> The statistics independent of twin laws show no twinning (more close to >>> untwinned than perfect twin). >>> Please suggest to me on how I proceed with refinement (twin OFF or ON). >>> >>> Thank you, >>> Venkat >>> >>> >>> >>> >>> >>> >>> >>> On Fri, Apr 5, 2024 at 1:00 AM Garib Murshudov >>> wrote: >>> >>>> Did you use twin refinement (is it really twin if you used that). >>>> If twin refinement was used then twin related intensities might have >>>> different resolution, in case when your crystal are pseudomerohedral >>>> twinned. >>>> >>>> Regards >>>> Garib >>>> >>>> On 4 Apr 2024, at 18:40, venkatareddy dadireddy >>>> wrote: >>>> >>>> CAUTION: This email originated from outside of the LMB: >>>> *.-owner-ccp...@jiscmail.ac.uk -.* >>>> Do not click links or open attachments unless you recognize the sender >>>> and know the content is safe. >>>> If you think this is a phishing email, please forward it to >>>> phish...@mrc-lmb.cam.ac.uk >>>> >>>> >>>> -- >>>> Hi Kay, >>>> >>>> Thank you very much for your insights. >>>> Following are the cell parameters from mtz and pdb header. >>>> *MTZ: 117.8560 66.1700 70.9040 90. 91.4240 90.000* >>>> >>>> *CRYST1 117.856 66.170 70.904 90.00 91.42 90.00 C 1 2 1* >>>> >>>> The only difference is in the 3rd decimal point. >>>> >>>> >>>> Thank you, >>>> Venkat >>>> >>>> >>>> >>>> On Tue, Apr 2, 2024 at 10:10 PM Kay Diederichs < >>>> kay.diederi...@uni-konstanz.de> wrote: >>>> >>>>> Hi Venkatareddy Dadireddy, >>>>> >>>>> do the unit cell parameters of your MTZ file and PDB file agree >>>>> exactly ? >>>>> >>>>> Take for example a cell of (100,110,120,90,90,90) in the header of the >>>>> MTZ file, >>>>> and (97,110,120,90,90,90) in the CRYST1 line of the PDB file. >>>>> >>>>> In this example, the (50,0,0) reflection would be at 2.0A resolution >>>>> if using the cell from the MTZ file, >>>
Re: [ccp4bb] Resolution discrepancy between MTZ file and Refinement
Thank you very much Garib. Venkat On Sat, Apr 6, 2024 at 1:12 AM Garib Murshudov wrote: > Unless you are confident that twin exists you should not use twin > refinement (Occam’s razor) > > > > On 5 Apr 2024, at 17:24, venkatareddy dadireddy > wrote: > > CAUTION: This email originated from outside of the LMB: > *.-owner-ccp...@jiscmail.ac.uk -.* > Do not click links or open attachments unless you recognize the sender and > know the content is safe. > If you think this is a phishing email, please forward it to > phish...@mrc-lmb.cam.ac.uk > > > -- > Hi Kay and Garib, > > Thank you for your input. > It is actually the twin refinement that gave rise to resolution > discrepancy. > For what reason I don't remember that I have turned the twin refinement ON > and the same job was cloned again and again. > With the twin refinement OFF, it gave rise to resolution present in the > MTZ (2.0 A). > From Xtriage: *the correlation between* > *the intensities related by the twin law 1/2*h-3/2*k, -1/2*h-1/2*k,-l with > an estimated twin* > *fraction of 0.10 is most likely due to an NCS axis parallel to the twin > axis*. > The statistics independent of twin laws show no twinning (more close to > untwinned than perfect twin). > Please suggest to me on how I proceed with refinement (twin OFF or ON). > > Thank you, > Venkat > > > > > > > > On Fri, Apr 5, 2024 at 1:00 AM Garib Murshudov > wrote: > >> Did you use twin refinement (is it really twin if you used that). >> If twin refinement was used then twin related intensities might have >> different resolution, in case when your crystal are pseudomerohedral >> twinned. >> >> Regards >> Garib >> >> On 4 Apr 2024, at 18:40, venkatareddy dadireddy >> wrote: >> >> CAUTION: This email originated from outside of the LMB: >> *.-owner-ccp...@jiscmail.ac.uk -.* >> Do not click links or open attachments unless you recognize the sender >> and know the content is safe. >> If you think this is a phishing email, please forward it to >> phish...@mrc-lmb.cam.ac.uk >> >> >> -- >> Hi Kay, >> >> Thank you very much for your insights. >> Following are the cell parameters from mtz and pdb header. >> *MTZ: 117.8560 66.1700 70.9040 90.0000 91.4240 90.000* >> >> *CRYST1 117.856 66.170 70.904 90.00 91.42 90.00 C 1 2 1* >> >> The only difference is in the 3rd decimal point. >> >> >> Thank you, >> Venkat >> >> >> >> On Tue, Apr 2, 2024 at 10:10 PM Kay Diederichs < >> kay.diederi...@uni-konstanz.de> wrote: >> >>> Hi Venkatareddy Dadireddy, >>> >>> do the unit cell parameters of your MTZ file and PDB file agree exactly ? >>> >>> Take for example a cell of (100,110,120,90,90,90) in the header of the >>> MTZ file, >>> and (97,110,120,90,90,90) in the CRYST1 line of the PDB file. >>> >>> In this example, the (50,0,0) reflection would be at 2.0A resolution if >>> using the cell from the MTZ file, >>> but it would be at 1.94A resolution if calculating the resolution based >>> on the cell from the PDB file. >>> >>> So perhaps REFMAC5 takes the cell from the PDB file, and phenix.refine >>> takes the cell from the MTZ? >>> I didn't check but it may be worth finding out. >>> >>> HTH, >>> Kay >>> >>> >>> On Tue, 2 Apr 2024 21:16:57 +0530, venkatareddy dadireddy < >>> venkatda...@gmail.com> wrote: >>> >>> >Hi, >>> > >>> >The resolution range in my MTZ file is 70.88 - 2.0 A. When I refined my >>> >structure using REFMAC5, the resolution that it gives is 70.88 - 1.94 A, >>> >the difference of 0.04 A. I also used Phenix.refine which gives the >>> >resolution output as it is in the MTZ file. Again, EDS (validation >>> report) >>> >gives the right resolution. What could be the possible reason for this >>> >discrepancy? I have the structure deposited in the Protein Data Bank >>> and it >>> >is on hold. Thank you in advance for your help. >>> > >>> >Thank you, >>> > >>> > >>> > >>> > >>> > >>> > >>> >*Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of >>> >Physics,IISc, Banglore.Cell: 07259492227* >>> > >>> >#### >>> > >>> >To unsubs
Re: [ccp4bb] Resolution discrepancy between MTZ file and Refinement
Hi Kay and Garib, Thank you for your input. It is actually the twin refinement that gave rise to resolution discrepancy. For what reason I don't remember that I have turned the twin refinement ON and the same job was cloned again and again. With the twin refinement OFF, it gave rise to resolution present in the MTZ (2.0 A). >From Xtriage: *the correlation between* *the intensities related by the twin law 1/2*h-3/2*k, -1/2*h-1/2*k,-l with an estimated twin* *fraction of 0.10 is most likely due to an NCS axis parallel to the twin axis*. The statistics independent of twin laws show no twinning (more close to untwinned than perfect twin). Please suggest to me on how I proceed with refinement (twin OFF or ON). Thank you, Venkat On Fri, Apr 5, 2024 at 1:00 AM Garib Murshudov wrote: > Did you use twin refinement (is it really twin if you used that). > If twin refinement was used then twin related intensities might have > different resolution, in case when your crystal are pseudomerohedral > twinned. > > Regards > Garib > > On 4 Apr 2024, at 18:40, venkatareddy dadireddy > wrote: > > CAUTION: This email originated from outside of the LMB: > *.-owner-ccp...@jiscmail.ac.uk -.* > Do not click links or open attachments unless you recognize the sender and > know the content is safe. > If you think this is a phishing email, please forward it to > phish...@mrc-lmb.cam.ac.uk > > > -- > Hi Kay, > > Thank you very much for your insights. > Following are the cell parameters from mtz and pdb header. > *MTZ: 117.8560 66.1700 70.9040 90. 91.4240 90.000* > > *CRYST1 117.856 66.170 70.904 90.00 91.42 90.00 C 1 2 1* > > The only difference is in the 3rd decimal point. > > > Thank you, > Venkat > > > > On Tue, Apr 2, 2024 at 10:10 PM Kay Diederichs < > kay.diederi...@uni-konstanz.de> wrote: > >> Hi Venkatareddy Dadireddy, >> >> do the unit cell parameters of your MTZ file and PDB file agree exactly ? >> >> Take for example a cell of (100,110,120,90,90,90) in the header of the >> MTZ file, >> and (97,110,120,90,90,90) in the CRYST1 line of the PDB file. >> >> In this example, the (50,0,0) reflection would be at 2.0A resolution if >> using the cell from the MTZ file, >> but it would be at 1.94A resolution if calculating the resolution based >> on the cell from the PDB file. >> >> So perhaps REFMAC5 takes the cell from the PDB file, and phenix.refine >> takes the cell from the MTZ? >> I didn't check but it may be worth finding out. >> >> HTH, >> Kay >> >> >> On Tue, 2 Apr 2024 21:16:57 +0530, venkatareddy dadireddy < >> venkatda...@gmail.com> wrote: >> >> >Hi, >> > >> >The resolution range in my MTZ file is 70.88 - 2.0 A. When I refined my >> >structure using REFMAC5, the resolution that it gives is 70.88 - 1.94 A, >> >the difference of 0.04 A. I also used Phenix.refine which gives the >> >resolution output as it is in the MTZ file. Again, EDS (validation >> report) >> >gives the right resolution. What could be the possible reason for this >> >discrepancy? I have the structure deposited in the Protein Data Bank and >> it >> >is on hold. Thank you in advance for your help. >> > >> >Thank you, >> > >> > >> > >> > >> > >> > >> >*Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of >> >Physics,IISc, Banglore.Cell: 07259492227* >> > >> > >> > >> >To unsubscribe from the CCP4BB list, click the following link: >> >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 >> > >> >This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a >> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are >> available at https://www.jiscmail.ac.uk/policyandsecurity/ >> > >> >> >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 >> >> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a >> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are >> available at https://www.jiscmail.ac.uk/policyandsecurity/ >> > > > -- > > > > > > > *Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of > Physics,IISc, Banglore.Cell: 07259492227* > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > -- *Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of Physics,IISc, Banglore.Cell: 07259492227* To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Resolution discrepancy between MTZ file and Refinement
Hi Kay, Thank you very much for your insights. Following are the cell parameters from mtz and pdb header. *MTZ: 117.8560 66.1700 70.9040 90. 91.4240 90.000* *CRYST1 117.856 66.170 70.904 90.00 91.42 90.00 C 1 2 1* The only difference is in the 3rd decimal point. Thank you, Venkat On Tue, Apr 2, 2024 at 10:10 PM Kay Diederichs < kay.diederi...@uni-konstanz.de> wrote: > Hi Venkatareddy Dadireddy, > > do the unit cell parameters of your MTZ file and PDB file agree exactly ? > > Take for example a cell of (100,110,120,90,90,90) in the header of the MTZ > file, > and (97,110,120,90,90,90) in the CRYST1 line of the PDB file. > > In this example, the (50,0,0) reflection would be at 2.0A resolution if > using the cell from the MTZ file, > but it would be at 1.94A resolution if calculating the resolution based on > the cell from the PDB file. > > So perhaps REFMAC5 takes the cell from the PDB file, and phenix.refine > takes the cell from the MTZ? > I didn't check but it may be worth finding out. > > HTH, > Kay > > > On Tue, 2 Apr 2024 21:16:57 +0530, venkatareddy dadireddy < > venkatda...@gmail.com> wrote: > > >Hi, > > > >The resolution range in my MTZ file is 70.88 - 2.0 A. When I refined my > >structure using REFMAC5, the resolution that it gives is 70.88 - 1.94 A, > >the difference of 0.04 A. I also used Phenix.refine which gives the > >resolution output as it is in the MTZ file. Again, EDS (validation report) > >gives the right resolution. What could be the possible reason for this > >discrepancy? I have the structure deposited in the Protein Data Bank and > it > >is on hold. Thank you in advance for your help. > > > >Thank you, > > > > > > > > > > > > > >*Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of > >Physics,IISc, Banglore.Cell: 07259492227* > > > > > > > >To unsubscribe from the CCP4BB list, click the following link: > >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > >This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > -- *Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of Physics,IISc, Banglore.Cell: 07259492227* To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Resolution discrepancy between MTZ file and Refinement
Hi, The resolution range in my MTZ file is 70.88 - 2.0 A. When I refined my structure using REFMAC5, the resolution that it gives is 70.88 - 1.94 A, the difference of 0.04 A. I also used Phenix.refine which gives the resolution output as it is in the MTZ file. Again, EDS (validation report) gives the right resolution. What could be the possible reason for this discrepancy? I have the structure deposited in the Protein Data Bank and it is on hold. Thank you in advance for your help. Thank you, *Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of Physics,IISc, Banglore.Cell: 07259492227* To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] DNA RNA annealing problem
Hi Weifei, We use SSC buffer for annealing oligos (we use DNA). That works pretty fine with us. Give a try. Composition is in below URL http://www.bioprotocols.info/reagent_and_buffer_recipes/20x-SSC.php Good Luck, Venkatareddy. On Fri, Jul 3, 2015 at 8:44 PM, ChenWeiFei weife...@outlook.com wrote: Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei -- *Venkatareddy Dadireddy,B1-10,Prof. S. Ramakumar's Lab,Dept. of Physics,IISc, Banglore.Cell: 07259492227*
[ccp4bb]
Thank you all for your suggestions. I try various methods suggested and hope they work. Regards Venkat On Wed, Jan 22, 2014 at 11:44 PM, Roger Rowlett rrowl...@colgate.eduwrote: Thrombin cleavage of His-tags seems to work pretty well in our hands, which includes mostly undergraduates for the hands-on work. I would encourage you to closely RTFM (read the friendly manual). Thrombin is not all that specific if used at the wrong (too high) concentration, so under the worst of conditions, it may chew up your protein pretty good. We have generally found that (following directions) a series of test cleavage reactions with the thrombin concentration serially diluted by say 10X is necessary to find the optimal thrombin concentration. Once the correct concentration is found, you can scale up. (We normally scale up to 2 mg protein, which we can do in 2 mL batches.) As the thrombin degrades, the appropriate dilution changes. Maybe 10,000-fold this week, maybe 1,000-fold next month. We have obtained really excellent results using thrombin, but don't do it often. So maybe we are just lucky. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 1/22/2014 1:46 AM, venkatareddy dadireddy wrote: Dear All, My protein is cloned in pET-15b vector, contains His- tag, thrombin cleavage site and extra sequence of 20 amino acids from vector. I crystallized without cleaving extra sequence and never got any crystal hits/crystals. Once, I tried thrombin reaction and it didn't work. Here I would like to know how efficient the cleavage by thrombin and it is kind of you, can provide protocol and buffer system for reaction. Thank you Venkat. -- *Venkatareddy Dadireddy, B1-10,Prof. S. Ramakumar's Lab, Dept. of Physics,IISc, Banglore. Cell: 07259492227*
[ccp4bb] DNA Protein co- Crystallization
Hi, I'm working on DNA binding protein, looking to co-crystallize protein- DNA complex and have no previous experience. Your suggestion would be very precious on the following queries. 1. My protein is 646 amino acid long and it exists as homodimer. It is also having around 20 amino acid extra sequence from vector. Will vector sequence affect crystallization? 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. 3. What is the length of DNA to be used? 4. What is purity of oligos to be used? Is it HPLC pure or normal desalted ones. I have read on CCP4 mails for screening purpose normal oligos are fine. Please comment on that. 3. Any other suggestions on Protein DNA co- crystallization. Thanks venkat
Re: [ccp4bb] DNA Protein co- Crystallization
Dear All, Thank you very much for your valuable suggestions. Venkat On Sat, Jan 4, 2014 at 9:53 AM, Acoot Brett acootbr...@yahoo.com wrote: Dear All, For the question, I think for a protein-DNA interaction, the protein may interact with any sequences of DNA, which will give a lot of combination of protein-DNA sequence for crystallization screening. Or do anyone regard to just try the crystallization of the protein-one specific sequence DNA fragment for the trial (for example the DNA sequence with the highest binding affinity)? In another word, does the easiness of the crystallization has relation with how strong the protein interacts with the DNA sequence? Acoot On Saturday, 4 January 2014 11:02 AM, rajakumara eerappa reera...@gmail.com wrote: My suggestions are 1 try complementary and non-complementary overhangs which can form Watson-crick and/ or Hoogstein base pairing. 2. If it binds self-complementary duplexes then try them also. 3. Peg conditions with slight acidic pH are more suitable. 4. Divalent cation salts (Ca, Mg, Mn) in crystallization. Wish you good luck Raj On Friday, January 3, 2014, venkatareddy dadireddy wrote: Hi, I'm working on DNA binding protein, looking to co-crystallize protein- DNA complex and have no previous experience. Your suggestion would be very precious on the following queries. 1. My protein is 646 amino acid long and it exists as homodimer. It is also having around 20 amino acid extra sequence from vector. Will vector sequence affect crystallization? 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. 3. What is the length of DNA to be used? 4. What is purity of oligos to be used? Is it HPLC pure or normal desalted ones. I have read on CCP4 mails for screening purpose normal oligos are fine. Please comment on that. 3. Any other suggestions on Protein DNA co- crystallization. Thanks venkat -- *Venkatareddy Dadireddy, B1-10,Prof. S. Ramakumar's Lab, Dept. of Physics,IISc, Banglore. Cell: 07259492227*
