[ccp4bb] Excellent Opportunity for Postdoc in Ion-Channel Structural Biology
Dear Structural Biology Scholars, I would like to bring your attention to an excellent opportunity to unravel the structure-function details of ion-channels as a postdoc. The Grosman lab at the University of Illinois at Urbana-Champaign is actively looking to fill a recent opening of NIH-funded postdoctoral position. The laboratory has an excellent record in combining high-resolution electrophysiology, molecular simulations, and structural biology to address fundamental questions about ion-channel mechanisms. Previous experience with the purification of eukaryotic membrane proteins is a plus and structure determination using single-particle cryo-EM is highly preferred. Salary commensurate with the experience level. The University of Illinois Urbana Champaign is an excellent place for research and life with an easy commute, lower-cost housing, easy access to all needs, and a vibrant community with easily accessible cities like Chicago, Indianapolis, St. Louis. Interested individuals should contact Prof. Claudio Grosman ( gros...@illinois.edu) with a CV, a 1–2 page description of previous research, and names and contact information of 2–3 references. Students in their final year of Ph.D. training are also welcome to apply. Recent select publications from the Grosman lab are listed below. A complete list of publications from the Grosman lab can be found on Prof. Claudio Grosman’s NCBI bibliography page ( https://www.ncbi.nlm.nih.gov/myncbi/claudio.grosman.1/bibliography/public <https://urldefense.com/v3/__https:/www.ncbi.nlm.nih.gov/myncbi/claudio.grosman.1/bibliography/public__;!!DZ3fjg!rQbPPU9HmgERY4wmiV22sCEFfhgBKyPAgmAjAc4NIwePpODucsYnvVtDcXV3NFUKog$> ). *Recent Select Publications * *· Kumar P, Cymes GD, Grosman C. Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel. Proc. Natl. Acad. Sci. USA. 2021;118(23). * *doi: 10.1073/pnas.2100164118 * *· Cymes GD, Grosman C. Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains. Proc. Natl. Acad. Sci. USA. 2021;118(14). doi: 10.1073/pnas.2021016118 * *doi: 10.1038/s41467-021-21680-9 * *· Kumar P, Wang Y, Zhang Z, Zhao Z, Cymes GD, Tajkhorshid E, Grosman C. Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer. Proc. Natl. Acad. Sci. USA. 2020;117(3):1788-1798. * *doi: 10.1073/pnas.1906823117 * -- Pramod Kumar, Ph.D. 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] Help to fight COVID-19
Dear Structure Biologists, We have some significant expertise in stabilizing the membrane proteins and domains in different kinds of membranous environments such as liposomes (small and giant vesicles suitable for biochemical and electrophysiological analysis), nanodiscs and malic acid based polymers. We are highly proficient for working up to BSL2 level experiments. In this tough time of COVID-19 pandemic we would love to share/help/collaborate with our expertise to any lab/organization interested in developing any kind of fundamental study or therapeutics. Please just contact us and we would do our level best. Pramod Kumar, Ph.D. Postdoctoral research associate, Grosman Lab Department of Molecular and Integrative Physiology University of Illinois at Urban-Champaign. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Interesting DNA contamination
Hi Thanks all, I appreciate all the valuable inputs.. Piush.. I ll be trying Benzonase up next.. but since the DNA appears so secluded for DNAses, it makes me little skeptical as mg and DNAse already been there for ON dialysis. Tim if the DNA binds to the protein, wouldn't this affect the interpretation of the Agarose gel? I can send you the Ni-elution SDS pic where the GFP taged protein and DNA band appers at two different different and distinct sizes. 500 bases should result in a distinct shift during gel filtration. Do you observe this? Since I don't have free protein to compare with DNA-bound, m not able to see the shift of peak. While waiting for suggestions, you might set up crystallization trials just in case? Yea, just did that :) .. Tom Why not crystallize the whole complex? It would be more interesting than the protein alone- higher impact and all that. kept the trays, wish me good luck :) Dan 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. Yes, Its nice piece of info. Just one concern eats me, that is the longer wash that I observed some time with mem-proteins may lead to release of native bound lipids and causes deterioration. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase breathing) to disrupt the interaction. I ll be doing that provided it tolerate. 3) Combine salt with low concentrations of denaturants. Incorporated to optimization list 4) Try a couple of different restriction enzymes such as DpnI or FatI to see if you can break it into smaller fragments. If you can, you maybe able to clone into a cloning vector with compatable ends/blunt ligation to sequence and identify the region of host DNA that is causing the problem. Excellent input, ll be doing. ... paul Stepwise addition to 1% PEI (polyethylenimine) following cell lysis (before dialysis) should do the trick. You mean, membrane and protein extraction as well Ni-Aff in presence of ~1% PEI? ... Katharina Since your protein apparently binds ATP – do you know the active site? Maybe its worthwhile to do an active site mutant (or any other possible DNA binding mutant?) and to see if it makes any difference in DNA binding? Nice input, Working on it. Another question is, if the DNA you observe is somehow an homogeneous species. Yes, consistantly Otherwise, maybe there’s a way to somehow digest the DNA further, in presence of the protein, after purification to obtain an homogeneous species, than to get rid of the nucleases again and than to simply set up crystallization trays. To set up crystalliyation trials I would do anyways, in case you have enough protein. Kept Trays, :) Thanks again :) Pramod
[ccp4bb] Interesting DNA contamination
Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
Re: [ccp4bb] str solving problem
Dear Eugene plz find the merging statics over this link https://www.dropbox.com/sh/3155bp0c8axo7tx/0P1RWTTD8z?n=21758536 I have tried different subset of images for indexing, only cell edges are changing very marginal ( 1 ) but no change in space group. Dear Manfred I have collected my data over mar345 detector, not present in detector type dropdown, how can I add. regards and thanks to all pramod On Mon, Jun 24, 2013 at 10:31 PM, Eugene Valkov eugene.val...@gmail.comwrote: Hi Pramod, Can you post your merging statistics in different space groups, not just log files from scaling? These are summarised nicely by Scala or Aimless. Also, have you tried indexing from different subsets of images? Perhaps there is a substantial contribution from a 'satellite' crystal in one orientation or crystal will be less split? I've had cases where I could not index properly if I had just used 0 and 90, but when I tried different subsets of images it worked. This is very easy to do in iMosflm. Andrew Leslie or other Mosflm developers, if they are reading this, might well be interested in looking at your images as they are currently interested in these kinds of problems with multiple lattices (see *Acta Cryst.* (2013). D*69*, 1195-1203) Eugene On 21 June 2013 21:58, Pramod Kumar pramod...@gmail.com wrote: Dear ... Francis Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices.. I have used both HKL2000 and mosflm giving the same results (although I have used manual selection of spots as a trial but results are identical). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group.. I have given several efforts to the XDS but its giving error data image of particular no. does not exist (initially it was saying 11th image than i change image range then it says 21st and so on) *kindly check my data collection profile and XDS.INP* file in attachment' Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. raising the crystals and struggle is on the peak... Dear Eugene plz find the attached scale log file, scaling table of mosflm When you index spots in Mosflm, do your predictions agree with the spots? plz see the snapshot of predicted spots.. Dear Eleanor Yes both the molecule are visible in the ASU. Dear Pozharski Balbes pipeline hitting extremely high marks when fed into Phaser while being complete nonsense (it's a 150kDa multi-domain protein and resulting domain arrangement made absolutely no sense). Refinement was stuck with high R-values and I sadly gave up on it for now. I suspected that refmac step included in the pipeline artificially shifts the model so that it conforms to Patterson map better, which results in high score in Phaser. My domain arrangement is as expected, two molecules in ASU. thanks and regards pramod On Thu, Jun 20, 2013 at 3:50 PM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: As others say - the Rfactors look pretty good for MR, mine usually start over 50% even with a better model and one hopes they then decrease.. But you say you took the Balbes model into phaser? and I think Balbes automatically runs cycles of refinement so any comment on R factors may not mean much. Have you found both molecules in the asymmetric unit? You only give LLG for one? Eleanor On 19 June 2013 17:44, Eugene Valkov eugene.val...@gmail.com wrote: Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.eduwrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared
Re: [ccp4bb] str solving problem
Dear Robert I have cross checked by running the gel and silver stain that confirms it is only the protein i am targeting. no exact hit was obtained for this cell parameter and SG, as it previously checked during balbes run... thanks and regards pramod On Wed, Jun 19, 2013 at 2:28 PM, Robert Esnouf rob...@strubi.ox.ac.ukwrote: Hi Pramod, You mention this is the limitation of my data that came from three month old crystal. If the crystal took that long to grow (did it?) then a crystallized (1) proteolytic fragment or (2) low level contaminant is quite likely. 1) If you have the crystal still you could try to redissolve it and run a gel 2) If it is the second case then the unit cell is probably already in the PDB. You can check with this tool... http://www.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi Good luck, Robert -- Dr. Robert Esnouf, University Research Lecturer and Head of Research Computing, Wellcome Trust Centre for Human Genetics, Old Road Campus, Roosevelt Drive, Oxford OX3 7BN, UK Emails: rob...@strubi.ox.ac.uk Tel: (+44) - 1865 - 287783 and rob...@well.ox.ac.uk Fax: (+44) - 1865 - 287547 Original message Date: Wed, 19 Jun 2013 03:20:57 +0530 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Pramod Kumar pramod...@gmail.com) Subject: Re: [ccp4bb] str solving problem To: CCP4BB@JISCMAIL.AC.UK Apology for delay Dear Abhinav.. Kept the ratio 2 with correlation of completeness above 75 r factor reduces with under -5 to 6 lees than r free. I am trying different models.. Dear Herman -Is the protein that crystallized the protein you think it is? Plasmids do get mixed-up and sometimes a more abundant natural protein with about the same molecular weight as the protein of interest gets purified. Yes, I have done silver stain that confirms exact MW, further related literature and biological properties supports, -How do the diffraction images look like: are there strong ice-rings, multiple diffraction patterns or other problems? Could there have been misindexing in one of the directions? no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) Dear Eugene the phaser .sol file states... SOLU SET RFZ=6.6 TFZ=8.8 PAK=0 LLG=102 TFZ==8.2 LLG=1804 TFZ==22.9 SOLU SPAC C 2 2 21 SOLU 6DIM ENSE ensemble1 EULER 180.061 0.000 0.000 FRAC -0.49950 -0.50021 -0.6 BFAC 0.0 SOLU ENSE ensemble1 VRMS 0.877 for building the model I used the 'autorikshaw pipeline' and it has given rfact/rfree 0.4645 / 0.4888 but back to refmac again starts rising Dear Edward Patterson analysis obtained from xtrige... Xtrige summary Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws I^2/I^2 : 2.078 F^2/F^2 : 0.777 |E^2-1| : 0.758 |L|, L^2: 0.507, 0.340 Multivariate Z score L-test: 2.003 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. No (pseudo)merohedral twin laws were found. Patterson analyses - Largest peak height : 4.674 (corresponding p value : 0.95872) The largest off-origin peak in the Patterson function is 4.67% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics behave as expected. No twinning is suspected. integration in P1 and attempt to carry out molecular replacement in P1 from this Zanuda concludes as follow ^ |5 | C 2 2 21 | 0.5985 | 0.4265 | 0.3918 | 0.4880 | - | 1 | P 1| 0.5308 | 0.4236 | 0.3970 | 0.5024 | | 2 | P 1 21 1 | 0.6302 |--| 0.3912 | 0.4989 | | 5 | C 2 2 21 | 0.7318 |--| 0.3804 | 0.5009 | - |5 | C 2 2 21 | 0.7318 |--| 0.3804 | 0.5009 | - R-factor in the original subgroup is NOT the best. The original spacegroup assignment seems to be incorrect. thanks and regards pramod On Tue, Jun 18, 2013 at 3:32 PM, Edward Lowe edward.l...@bioch.ox.ac.uk wrote: Dear Pramod, It's difficult to be certain based on what you have posted but my best guess would be problems
[ccp4bb] str solving problem
Dear group I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO, now the model for molecular replacement with closest identity of 31 given a contrast of 2, score 0.30 and wrfac 0.60. but balbes uses different models with lesser identity, no matter which way I am going the rFree keep on increasing during refinement in refmac, when I build the model in coot with deletion and addition of residue it starts with relatively low but gradually rises through almost all cycles although model fits to the density well and residue are building, coot validation parameters are also reasonable OK for geometry, rotamer, density fit,.. now my question * where should i first check for possible correction? * In molecular replacement what should be the red line for identity and related criteria? * if initial rFree starts around 50, how likely that its not the right way? * my rms bond angle is close to 1 while the bond length is 0.01 and chiral 0.1 concludes what is serious? *sincere apology for amateur query* if any... thanks in advance pramod -- Pramod Kumar. Graduate Student. Crystallography lab. Department Of Biotechnology. Indian Institute Of Technology Roorkee Uttranchal.247667 India +919359189657.
Re: [ccp4bb] str solving problem
*Dear Abhinav Kumar * *thanks for kind suggestions * * I have tried as follow. 1. You should try to identify the correct space group first. *integration in p21 given the following statics in pointless * * Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation* * [h,k,l] 0.5660.9570.094 25580 0.00* * [l,-k,h] 0.4340.8530.195 25580 0.20 * * *integration in c2221 given the following statics in pointless * * Spacegroup TotProb SysAbsProb Reindex Conditions* * * * ( 20)0.997 0.997 00l: l=2n (zone 1) * * moreover Zanuda and molrep's chekall space group option also suggests c2221, 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success.Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? * * * this is the limitation of my data that came from three month old crystal, there are two molecules in ASU (what is the extant to say reasonably good please ?), initially phaser was failed, with the balbes output as ensemble it worked with following profile .. * *** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 *** * * * Current is Best Solution (first)* * New Best LLG : 101.7 (8.70)* * Best Search Component so far: ensemble1 * * * *** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING 2.5.2 *** * * * Resolution of All Data (Number):2.91 47.64 (15877)* * Resolution of Selected Data (Number): 2.91 47.64 (15877)* * * * * * Refinement Table (Unsorted)* * ---* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YES C 2 2 21 * * * * * * Refinement Table (Sorted)* * -* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YES C 2 2 21 **but again the refmac is showing * * *R factor 0.4404 0.4203 R free 0.4955 0.5027 Rms BondLength 0.0254 0.0093 Rms BondAngle 3.1234 1.4581 Rms ChirVolume 0.1741 0.0706 * * * * * * *3. Did you check for twinning? * *TWINNING SUMMARY* * * *Twinning fraction from H-test: 0.00* *L-statistic from L-Test: 0.48* * * * Relation between L statistics and twinning fraction:* * Twinning fraction = 0.000 L statistics = 0.500:* * Twinning fraction = 0.100 L statistics = 0.440:* * Twinning fraction = 0.500 L statistics = 0.375:* *NO Twinning detected* * * * * *thanks and regards * * pramod... * * * * * * * * * *On Mon, Jun 17, 2013 at 10:15 PM, Abhinav Kumar abhin...@slac.stanford.edu wrote: * *Hi Pramod, 1. You should try to identify the correct space group first. Did you integrate in p1 and run pointless? ** 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success. Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? 3. Did you check for twinning? * * Thanks, Abhinav * * JCSG@SSRL, SLAC (650) 926-2992 * * On 06/17/2013 09:35 AM, Pramod Kumar wrote: * *Dear group * * I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO, now the model for molecular replacement with closest identity of 31 given a contrast of 2, score 0.30 and wrfac 0.60. but balbes uses different models with lesser identity, no matter which way I am going the rFree keep on increasing during refinement in refmac, when I build the model in coot with deletion and addition of residue it starts with relatively low but gradually rises through almost all cycles although model fits to the density well and residue are building, coot validation parameters are also reasonable OK for geometry, rotamer, density fit,.. ** * *now my question * ** where should i first check for possible correction? * ** In molecular replacement what should be the red line for identity and related criteria? * ** if initial rFree starts around 50, how likely that its not the right way? * ** my rms bond angle is close to 1 while the bond length is 0.01 and chiral 0.1 concludes what is serious
[ccp4bb] ccp4 start
Dear all I am facing problem with my installed ccp4 suit in opening through konsol. It is displaying a warning note as following * WARNING ** The directory /home/programs/tmp (assigned to CCP4_MASTER) does not exist. The CCP4 programs will not run correctly, and any installation attempt will have errors or will fail. * WARNING ** kindly give me suggestion to fix it, in simple steps. thanks in advance. -- Pramod Kumar. Graduate Student. Crystallography lab. Department Of Biotechnology. Indian Institute Of Technology Roorkee Uttranchal.247667 India +919359189657.
