Re: [ccp4bb] solve structure by molecular replacement
Your molecule seems to be titled towards at least one of the axis, so there's no problem with your dimensions in P21. Jürgen On Mar 7, 2010, at 7:40 PM, Chunmin L wrote: > Hi Jürgen, > > Many thanks for your message. Somehow Mosflm in CCP4 did not > recognize the images. Will try XDS. or d*TREK. Still no answers for > that P21 one-molecule solution. It does not collide with itself. The > model is about 60 A wide and 82 A long. Unit cell dimensions of a=50 > b=51 c=252. if we have 82 A along the 252A, the 60 A is wider than > the > other two dimensions. I was surprised to see the previous postdoc got > pretty good density for this molecule. > > Many thanks, > Chunmin > > On Sun, Mar 7, 2010 at 3:00 PM, Jürgen Bosch wrote: >> >> On Mar 7, 2010, at 4:35 PM, Chunmin L wrote: >> >> I can not reprocess the data to >> check the systematic absence to determine the space group (I do not >> have the right software to reprocess the raw image at this time). >> >> What exactly do you mean by that ? The world does not consist of HKL alone, >> there are other options available even CCP4 ones e.g. Mosflm or non-CCP4 >> e.g. XDS. or d*TREK >> Jürgen >> - >> Jürgen Bosch >> Johns Hopkins Bloomberg School of Public Health >> Department of Biochemistry & Molecular Biology >> Johns Hopkins Malaria Research Institute >> 615 North Wolfe Street, W8708 >> Baltimore, MD 21205 >> Phone: +1-410-614-4742 >> Lab: +1-410-614-4894 >> Fax: +1-410-955-3655 >> http://web.mac.com/bosch_lab/ >> - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] anomalous phase extension to native data
DM ? Can you use NCS ? Jürgen On Mar 7, 2010, at 6:43 PM, Stuart Endo-Streeter wrote: > Fellow crystallographers, > > I am helping a college with a structure and we've run into a bit of a > jam. She has a 2.5 ang sel-met dataset, solved, and a 1.8 ang native > dataset. We would like to extend the phase solution from the anomalous > data to the native data. Unfortunately I have not been able to figure > out how to do this using CCP4. The phase solution was calculated using > PHENIX, which I have no real experience with yet, having worked with > SHELX, CNS, and CCP4 in the past. Can anyone point out the programs, if > any, in CCP4 (v6.1.3) to extend the phases from the 2.5 anomalous to the > 1.8 native data? > > Thanks, > Stuart Endo-Streeter - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] solve structure by molecular replacement
Hi Jürgen, Many thanks for your message. Somehow Mosflm in CCP4 did not recognize the images. Will try XDS. or d*TREK. Still no answers for that P21 one-molecule solution. It does not collide with itself. The model is about 60 A wide and 82 A long. Unit cell dimensions of a=50 b=51 c=252. if we have 82 A along the 252A, the 60 A is wider than the other two dimensions. I was surprised to see the previous postdoc got pretty good density for this molecule. Many thanks, Chunmin On Sun, Mar 7, 2010 at 3:00 PM, Jürgen Bosch wrote: > > On Mar 7, 2010, at 4:35 PM, Chunmin L wrote: > > I can not reprocess the data to > check the systematic absence to determine the space group (I do not > have the right software to reprocess the raw image at this time). > > What exactly do you mean by that ? The world does not consist of HKL alone, > there are other options available even CCP4 ones e.g. Mosflm or non-CCP4 > e.g. XDS. or d*TREK > Jürgen > - > Jürgen Bosch > Johns Hopkins Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Phone: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-3655 > http://web.mac.com/bosch_lab/ >
[ccp4bb] anomalous phase extension to native data
Fellow crystallographers, I am helping a college with a structure and we've run into a bit of a jam. She has a 2.5 ang sel-met dataset, solved, and a 1.8 ang native dataset. We would like to extend the phase solution from the anomalous data to the native data. Unfortunately I have not been able to figure out how to do this using CCP4. The phase solution was calculated using PHENIX, which I have no real experience with yet, having worked with SHELX, CNS, and CCP4 in the past. Can anyone point out the programs, if any, in CCP4 (v6.1.3) to extend the phases from the 2.5 anomalous to the 1.8 native data? Thanks, Stuart Endo-Streeter
Re: [ccp4bb] solve structure by molecular replacement
On Sunday 07 March 2010, Jürgen Bosch wrote: > > On Mar 7, 2010, at 4:35 PM, Chunmin L wrote: > > I can not reprocess the data to > > check the systematic absence to determine the space group (I do not > > have the right software to reprocess the raw image at this time). > > What exactly do you mean by that ? The world does not consist of HKL alone, > there are other options available even CCP4 ones e.g. Mosflm or non-CCP4 e.g. > XDS. or d*TREK And anyhow, you don't need to check the systematic absences. You need to check whether there are or are not additional 2-fold axes. Have the program "pointless" take a look at your P21 data set. Ethan
Re: [ccp4bb] solve structure by molecular replacement
On Mar 7, 2010, at 4:35 PM, Chunmin L wrote: > I can not reprocess the data to > check the systematic absence to determine the space group (I do not > have the right software to reprocess the raw image at this time). What exactly do you mean by that ? The world does not consist of HKL alone, there are other options available even CCP4 ones e.g. Mosflm or non-CCP4 e.g. XDS. or d*TREK Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] solve structure by molecular replacement
Hi Dr. Merritt, Thank you very much for your reply. >> We are trying to solve a protein structure by molecular replacement. >> The protein crystal has unit cell dimensions of a=50 b=51 c=252, >> alpha=90 beta=90.4 gamma=90. > >> We processed the data both as P21 (two molecules in A. U.) > > OK > >> and P21212 (one molecule in A. U.). > > What about the other possible orthorhombic spacegroups? > You need to try at least > P212121 > P22121 > P21221 > You should at the least try that 3rd one, since it is the equivalent > to your P21 setting. I am planning to do that. Did you notice that the model molecule is fatter than my unit cell but we still be able to obtain good density for one molecule in the P21 setting? I can not reprocess the data to check the systematic absence to determine the space group (I do not have the right software to reprocess the raw image at this time). But P21 and P21212 gave Rmerge around 10% up to 2.5 A resolution. I checked with the lab members, about three postdoc fellows worked on this project before, I was told the space group was sure P21212. >> We have built a model based on sequence homology. >> It is very interesting that we >> still found one molecule using P21 data, reasonably GOOD density >> although the model is about 60 A wide and 82 A long (just refine this >> one molecule using P21 data, the Overall R factor=0.4731, Overall >> figure of merit=0.4939, the protein molecule seems have no symmetry by >> itself). > > Wait. > So your P21 solution only contains one molecule of the two that are expected? > If you can't fit a second molecule in P21, then I don't see how moving to > a higher-symmetry space group can work. I am sorry my message may not that clear. We failed P21212 then tried P21. >> We can not find any solution using P21212. The protein >> seems have three domains based on our model. We tried to using only >> one of the domains. The results were similar. Any suggestions as to >> what else is possibly wrong except that we have not found the correct >> solution? > > I suggest not to use a homology model for molecular replacement. > Better to use the homologous structure[s] directly, without modifying > their coordinates. See recent thread. Thanks for the good suggestions. Chunmin
Re: [ccp4bb] solve structure by molecular replacement
On Sunday 07 March 2010, Ethan Merritt wrote: > On Sunday 07 March 2010, Chunmin L wrote: > > Dear All, > > > > We are trying to solve a protein structure by molecular replacement. > > The protein crystal has unit cell dimensions of a=50 b=51 c=252, > > alpha=90 beta=90.4 gamma=90. > > > We processed the data both as P21 (two molecules in A. U.) > > OK > > > and P21212 (one molecule in A. U.). > > What about the other possible orthorhombic spacegroups? > You need to try at least >P212121 >P22121 >P21221 > You should at the least try that 3rd one, since it is the equivalent > to your P21 setting. Oops. I meant to say that the symmetry of the 3rd one is _not_ a superset of your P21 setting.
Re: [ccp4bb] solve structure by molecular replacement
On Sunday 07 March 2010, Chunmin L wrote: > Dear All, > > We are trying to solve a protein structure by molecular replacement. > The protein crystal has unit cell dimensions of a=50 b=51 c=252, > alpha=90 beta=90.4 gamma=90. > We processed the data both as P21 (two molecules in A. U.) OK > and P21212 (one molecule in A. U.). What about the other possible orthorhombic spacegroups? You need to try at least P212121 P22121 P21221 You should at the least try that 3rd one, since it is the equivalent to your P21 setting. > We have built a model based on sequence homology. > It is very interesting that we > still found one molecule using P21 data, reasonably GOOD density > although the model is about 60 A wide and 82 A long (just refine this > one molecule using P21 data, the Overall R factor=0.4731, Overall > figure of merit=0.4939, the protein molecule seems have no symmetry by > itself). Wait. So your P21 solution only contains one molecule of the two that are expected? If you can't fit a second molecule in P21, then I don't see how moving to a higher-symmetry space group can work. > We can not find any solution using P21212. The protein > seems have three domains based on our model. We tried to using only > one of the domains. The results were similar. Any suggestions as to > what else is possibly wrong except that we have not found the correct > solution? I suggest not to use a homology model for molecular replacement. Better to use the homologous structure[s] directly, without modifying their coordinates. See recent thread.
Re: [ccp4bb] solve structure by molecular replacement
Dear All, We are trying to solve a protein structure by molecular replacement. The protein crystal has unit cell dimensions of a=50 b=51 c=252, alpha=90 beta=90.4 gamma=90. We processed the data both as P21 (two molecules in A. U.) and P21212 (one molecule in A. U.). We have built a model based on sequence homology. It is very interesting that we still found one molecule using P21 data, reasonably GOOD density although the model is about 60 A wide and 82 A long (just refine this one molecule using P21 data, the Overall R factor=0.4731, Overall figure of merit=0.4939, the protein molecule seems have no symmetry by itself). We can not find any solution using P21212. The protein seems have three domains based on our model. We tried to using only one of the domains. The results were similar. Any suggestions as to what else is possibly wrong except that we have not found the correct solution? Thanks in advance, Chunmin
[ccp4bb] Postdoctoral Position in Membrane Protein Biology at NINDS/NIH
*NINDS/NIH Postdoctoral Position in Membrane Protein Biology* Applications are invited for a postdoctoral position in the Membrane Protein Structure and Function Unit, National Institute of Neurological Disorders and Stroke, to work on the structure determination and functional aspects of G-protein-coupled receptors. Candidates will ideally have experience with membrane protein expression, purification, characterization and/or crystallization, but highly qualified candidates from other areas of biochemistry and structural biology are also encouraged to apply. Applicants must have a Ph.D. and fewer than 5 years of postdoctoral experience. Applicants should send their CV, and the names and addresses of three referees to: Reinhard Grisshammer MPSFU, National Institute of Neurological Disorders and Stroke, National Institutes of Health 5625 Fishers Lane, Room 4S12, Rockville MD 20852, USA E-mail: rkgr...@helix.nih.gov The NIH is dedicated to building a diverse community in its training and employment programs. -- Reinhard Grisshammer NINDS, NIH 5625 Fishers Lane, room 4S-12 Rockville, MD 20852 telephone +1 301 594 9223 fax +1 301-480-3934 email rkgr...@helix.nih.gov http://neuroscience.nih.gov/Lab.asp?Org_ID=541
Re: [ccp4bb] Reg Protein purification
Dear Sivaraman, in case no one mentioned it, i would simply try ion exchange. DNA will bind very strongly to anion exchange columns (obviously). (and probably your protein is basic?) worked for me many times. and note that if you have a smear on agarose only tells about the size distr. of your nucleic acid not protein. with regards to IMAC, to my understanding its not the imidazole but leaking metal ions that cause the precipitation, this has been discussed many times... (while of course you are better of changing the buffer, not a good idea to keep it in high imidazole) HTH, Tommi On Mar 6, 2010, at 7:00 PM, Chun Luo wrote: Hi Sivaraman, NAD+ has A259 peak absorbance. So you may not have that much nucleic acids contamination. However, it is not uncommon to have large amount of nucleic acids eluted from Ni columns. Adding our TurboNuclease (http://www.accelagen.com/TurboNuclease-protocol.htm ) in lysis buffer can significantly reduce nucleic acids contamination and lysate viscosity. TurboNuclease has thousands fold higher specific activity than DNaseI. So you only need a little bit. Many proteins aggregate/precipitate in imidazole. It’s a useful precaution to remove imidazole before concentrating proteins. You probably concentrated the protein to a minute volume for a 16/60 column and the concentration could be too high. Try desalting before concentrating the protein and try SEC at lower protein concentration. Good luck! Chun From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sivaraman Padavattan Sent: Saturday, March 06, 2010 4:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reg Protein purification Dear All, We are trying to purify an enzyme, which requires the co-factor NAD+ during catalysis by affinity column (Ni-NTA). After induction, the bacterial cells were harvested and lysed with 20 mM Tris pH 7.2, 500 mM NaCl, 5% glycerol, 5 MM B-ME. The resultant supernatant was passed through Ni-NTA and bound protein eluted with increasing concentration of Imidazole. The eluted proteins was concentrated and load onto gelfiltration (Superdex S-75 16/60) column. Our protein eluted as a aggregate along with other protein, where A260 was much greater than A280, indicative of large fraction of nucleic acid contamination. The eluant also appeared as a smear on 1% agarose gel electrophoresis. We introduced 1M NaCl in the lysis buffer to prevent the nucleic acid interaction. But most of our protein went in pellet after cell lysis. We look forward to your valuable suggestion to purify the protein free of nucleic acid contamination. Thanks in advance, Sivaraman Padavattan Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Reg Protein purification
Hi Sivaraman, One efficient way to remove nonspecifically associated DNA is to do polyethyleneimine (PEI) precipitation. Protocols are available online. Search for PEI precipitation. Also look up this reference for RNA polymerase purification: Burgess and Jendrisak. 1975 Biochemistry 14(21):4624-8 It is a great method when many others fail to efficiently remove DNA. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University On Mar 6, 2010, at 7:23 AM, Sivaraman Padavattan wrote: Dear All, We are trying to purify an enzyme, which requires the co-factor NAD+ during catalysis by affinity column (Ni-NTA). After induction, the bacterial cells were harvested and lysed with 20 mM Tris pH 7.2, 500 mM NaCl, 5% glycerol, 5 MM B-ME. The resultant supernatant was passed through Ni-NTA and bound protein eluted with increasing concentration of Imidazole. The eluted proteins was concentrated and load onto gelfiltration (Superdex S-75 16/60) column. Our protein eluted as a aggregate along with other protein, where A260 was much greater than A280, indicative of large fraction of nucleic acid contamination. The eluant also appeared as a smear on 1% agarose gel electrophoresis. We introduced 1M NaCl in the lysis buffer to prevent the nucleic acid interaction. But most of our protein went in pellet after cell lysis. We look forward to your valuable suggestion to purify the protein free of nucleic acid contamination. Thanks in advance, Sivaraman Padavattan
[ccp4bb] Cryoprotective ability of low molecular weight (400kDa) poly-gsmms-glutamic acid (PGA) polymer
Dear All Please can you tell me what is the Cryoprotective ability of low molecular weight (400kDa) poly-gamma-glutamic acid (LM-PGA) polymer. If it is cryoprotective then can anybody mail me the percentage of glycerol/EG addition for the given percentage of LM-PGA. Thanking you raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your preferred Email name! Now you can @ymail.com and @rocketmail.com. http://mail.promotions.yahoo.com/newdomains/aa/
