Re: [ccp4bb] Opinion on automation

Dear theresa xia2 does a great job so it can be used as your reference when processing your data manually, either for learning or improvement. When comparing different processing programs and protocols try to judge data quality by some objectice criteria like HA site identification, anomalous p

Re: [ccp4bb] Bicelle Crystallization in a Gryphon Robot

Sorry for self promotion but you might also consider the HILIDE method which is very similar to bicelle and sponge, only having fully solubilized protein:lipid:detergent complexes as input sample and therefore being fully compatible with regular liquid handling robotics. See Gourdon P et al 2011

Re: [ccp4bb] Crystal Structures as Snapshots

Another good lesson here: 2. The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure. Vestergaard B, Sanyal S, Roessle M, Mora L, Buckingham RH, Kastrup JS, Gajhede M, Svergun DI, Ehrenberg M. Mol Cell. 2005 Dec 22;20(6):929-3

Re: [ccp4bb] No diffraction

But first of all: try to add a synchrotron to the crystals Poul On 26/01/2012, at 16.48, Katherine Sippel wrote: > Might I suggest consulting the CCP4 user community wiki on the topic: > > http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality > > Good luck, > >

Re: [ccp4bb] detect dsDNA

We chewed pills with EtBr as kids in school to see if we brushed our teeth well - red colour on the edges, bad boy Poul On 01/10/2011, at 19.12, Jacob Keller wrote: > I actually looked at an EtBr MSDS a while ago, and was shocked at how > benign it was. I also heard from someone that they us

Re: [ccp4bb] drops swelling

Yes - add glycerol to the reservoir - 15% glycerol has a huge influence on the vapour diffusion gradients Poul On 09/09/2011, at 13.22, anita p wrote: > Dear Crystallographers, > I have set hanging drop trays with 2ul of protein and 2 ul of resorvior > solution. I have seen in some cases the

Re: [ccp4bb] Is there a protein that it could interact with different proteins by the same part?

Calmodulin Poul On 23/08/2011, at 16.55, Pete Meyer wrote: >> We know one protein can interact with different partners by different >> domains or different parts. Is there a protein that it could interact >> with different proteins by the same part (maybe the same part but in >> different conform

Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

Deng, You need a good synchrotron source with a low point spread detector, but not least a careful consideration in crystal mounting so that your crystal rotates apprx around the long axis during data collection to optimise the separation of reflections on the detector besides checking that

Re: [ccp4bb] MR with MD trajectories

Dear Wandu, It might be smeared density because it is missing from your phasing model, so no reason to reinvent the wheel with MD and Phaser. You see electron density features for the domain and you know its basic structure. You can place the domain manually - it would take you five minutes and

Re: [ccp4bb] How to crystallize a helicase

Dear Art, It seems twisted, indeed. First of all you need to verify that your protein is at all in a good state (you seem to imply that it is not with your remark on protein concentration). Basic biophyscial characterisation for stability and structural homogeniety is required to reach proper buf

Re: [ccp4bb] optimization of dumbbell like crystal

Dear harvey Consider your purification and preparation protocols - is the sample pure and homogenous? Poul On 21/02/2011, at 03.29, Harvey Rodriguez wrote: > Dear CCP4BBers, > > Recently I got a crystal which appeared in the condition 2.1M DL-Malic Acid, > pH 7.0. The crystal looks like a d

Re: [ccp4bb] Merging data to increase multiplicity

Not sure if you ever got an answer to the original question (but I have had some mail mishabs today, so perhaps some messages are missing in my thread) Anyway: for sure NO - you cannot process the data with different parameter settings and then merge the different integrations as a way to gain in

Re: [ccp4bb] Citations in supplementary material

I agree with Tassos - and don't deny the power of evolution. Choice of journals, submissions, peer review, editorial assistance and citations are all in our hands - publishing has become what we have made it. We can argue that we want it differently (and to stay in the evolution terms: I also kn

Re: [ccp4bb] DNA-protein complex.

Dear dengzq1987 (Gmail Research Institute?) You could use ammonium acetate as the solubilizing salt. It evaporates as ammonia and acetic acid, so that the ionic strength is gradually reduced by vapor diffusion. You would then have e.g. PEG in the reservoir and added to the drop, and you could a

Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)

Two separate crystals, but very similar data collection strategies P On 04/10/2010, at 21.48, Jacob Keller wrote: > - Original Message - From: "Poul Nissen" > To: > Sent: Monday, October 04, 2010 2:21 PM > Subject: Re: [ccp4bb] Radiation damage with crystals co

Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)

[1] the signal from Ta6Br12 is enormous and one will typically focus on low resolution (below 7 Å) so radiation sensitivity can be handled by a fairly low dose data collection We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth JP et al. 2007) and found that although we g

Re: [ccp4bb] R factor & R free struck

Try a lower symmetry, e.g. P21 or P1 with one or two octamers in the asymmetric unit (well, unit cell for P1), but you already know your packing so no problem solving it. It can sometimes be very subtle and your model refinement will be the most sensitive test for the correct space group - so do

Re: [ccp4bb] Introducing PDBprints - salient, at-a-glance info about PDB entries

Also road signs can be cleverly replaced <><> On 15/07/2010, at 16.40, Phoebe Rice wrote: > What would be wrong with WORDS? They were such a clever > invention. I can tell the difference between colors, but it > takes a second step to figure out what they mean anyway. Why > not just write "no

Re: [ccp4bb] metal-chelating affinity chromatography and FosCholine detergents

Dear Pascal, There can be a number of reasons for this. Maybe fos-cholines are not very well suited for your membrane proteins of interest? - have you checked for activity or aggregation and compared to other detergents? If the detergent is optimal you may consider moving/extending the tag or c

Re: [ccp4bb] Off-topic: crystallization after refolding

Try first to give it a shot and see if you get crystals without too much hassle and expense. I would recommend a size exclusion chromatography step after refolding and from that collect a uniform sample for crystallization. Poul On 12/07/2010, at 13.39, meindert lamers wrote: > Dear all, > >

Re: [ccp4bb] A strange case of MR

You mention that your Rsym is 0.6 - this seems outrageously high (except if the 0.6 is just for your outer resolution bin). If 0.6 is indeed the overall Rsym you have a basic problem of unit cell and space group assignment to reconsider. Check if your processing accounts for all spots and check

Re: [ccp4bb] DATA COLLECTION?

collection around absorption edges. You cannot do crystallography and get and evaluate proper results without studying the basic theory and practice - crystallography is science after all, not black-box analysis. Poul Poul Nissen, professor

Re: [ccp4bb] How to distinguish microcrystalline, quasicystalline and precipitation?

Whether the shiny precipitate can be redissolved or not is an important observation. If not: bad. If yes: good Also one can check if the shiny precipitate can be used for streak-seeding in new drops. If not: bad/neutral. It yes: good. A bad shiny precipitate also often forms a large connected s

Re: [ccp4bb] measuring volume of cavity

If you are down to a scale where you consider a single water molecule I would say that the volume is not a very useful parameter. I woudl rather ask if it makes chemical sense to place a water molecule, and if the structure is determined at a sufficient level of precision that you can ident

Re: [ccp4bb] Rsym problems...maybe???

I very much agree - refinement will tell you if the high-res data make sense. Another very good test is the Wilson plot - it should look straight and reasonable. Inflated I/sigI values will not escape a strange appearance such as the WIlson plot flattening out at higher resolution. I normal

Re: [ccp4bb] Expression of a protein of 43KDa

EF-Tu Poul On 18/02/2010, at 18.49, Armando Albert de la Cruz wrote: I am trying to overexpress a His-tagged protein of 29KDa in E.coli (BL21-codon plus) and I end up with a highly expressed product that of 43KDa that binds to the Ni-column. I also have nice crystals. Does anyone have any

[ccp4bb] Fwd: [ccp4bb] electron density slabs

This could be due to biscuit contamination of your protein - did you eat cake before setting up your crystallisation drops?Alternatively it could be a Fourier ripple - a very strong 5.4 Å resolution 00l reflection which is not correctPoulBegin forwarded message:From: hayato Jumonji

Re: [ccp4bb] Histogram matching in DM - question

Hi Peter, Histogram matching works well for RNA structure, but be aware that density modification in general can be very tricky at low resolution. If by any means you can exploit averaging, e.g. multicrystal averaging of non-isomorphous crystals, it will probably be very helpful. It will

Re: [ccp4bb] model bias

Great that you have MR phases - it will help you identify heavy-atom sites for phasing and perhaps even the sulphur sites will be enough. Poul On 11/08/2009, at 20.33, ManojSaxena wrote: Hi all, I am working with a protein that have 28% similar to my MR template. I have processed data in HKL

Re: [ccp4bb] coupling between occupancy and b-values in refinement

If there is serious doubt of a full occupancy of the ligand and it is of importance for the interpreation it can in fact be handled and even at lower resolution (we have done it 2.8 Å resolution for PDB 2C8K for example) - I'm not the referee but maybe he/she is right ;-) You need not refine

Re: [ccp4bb] Hanging vs. Sitting

We often find results to be very different between hanging and sitting drops (equilibration kinetics for one may be the explanation). Then there's the good thing of hanging drops that crystals rarely stick to the surface of the support facilitating the mounting procedure, in particular for

Re: [ccp4bb] DM and MR

..and I may add that for DMMULTI two "rather nonisomorphous" data sets of the same crystal form can work like a dream, in particular at low resolution where solvent flattening and histogram matching can be tricky, see for example Morth et al. 2007, Nature 450, 1043 & Pedersen et al. 200

Re: [ccp4bb] Could I improve an inconsecutive experimental map?

Hi alphar~ Check the sites you get from anomalous difference Fouriers and stick to those as your correct HA sites. At the same time you may consider not to refine the HA substructure on the anomalous differences but merely using the isomorphous differences. Maybe you need to change the sca

Re: [ccp4bb] LDAO & SDS-PAGE

Besides the K-SDS precipitation it can also be the problem of membrane proteins generally having a tendency to aggregate when boiled with SDS. Try to just add the Laemmli buffer and load the gel without boiling. This is the general procedure for membrane proteins. Poul On 18/12/2008, at 0

Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

...and in absence of TM domains and the 2D restriction of the membrane they will probably not dimerize as free domains in solution now suddenly gained the freedom of 3D diffusion On 11/12/2008, at 19.03, Nathaniel Echols wrote: On Thu, Dec 11, 2008 at 8:09 AM, Santarsiero, Bernard D. wrote

Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

I would say that all crystals represent hyper-oligomeric structures, but never mind, I know what you mean ;-) the E. coli EF-Tu:EF-Ts complex is a good example - the structure clearly indicates an (EF-Tu:EF-Ts)2 dimer, and the T. thermophilus EF- Tu:EF-Ts is even a disulphide-linked dimer. H

Re: [ccp4bb] Detergent Crystals In Tube?

The carry-over would be DDM, and my guess would be DDM crystals formed upon dehydration. The DDM concentration will be huge after repeated dilution/concentration cycles. DDM micelles will hardly pass the 50 kDa cut-off filter so imagine how much you have added! Poul On 31/10/2008, at 02.21,

Re: [ccp4bb] asymmetric oligomers

been approximately located. ====Poul Nissen, professor, ph.d.Centre for Membrane Pumps in Cells and Disease - PUMPKIN& Centre for Structural BiologyUniversity of Aarhus, Dept. Molecular BiologyGustav Wieds Vej 10C, DK - 8000 Aarhus C, De

Re: [ccp4bb] Protein concentration

Often the exact concentration is not so important compared to the ability to establish proportional read-out, ease and reproducibility so that systematic variations and comparisons can be made. For considerations of molar ratios of for example protein:ligand complexes one would often screen

Re: [ccp4bb] about anisotrophic diffraction

Ji, probably the oil doesn't work and the glycerol gets you into problems with phase separation in 2.5 M AmS I recommend sucrose or other sugars with AmS - 20-22% (w/vol) should do and you get no phase separation At the same time you may need to increase the AmS considerably - often the pro

Re: [ccp4bb] which concentrated salt has lowest vapour pressure?

We normally use AmS or glycerol. Poul On 07/04/2008, at 15.46, Kay Diederichs wrote: Dear all, a protein which we work on is available in low quantity. The only crystallization screen we set up is completely clear, no precipitate, nothing. Now we would like to modify the reservoirs of th

Re: [ccp4bb] twinned?

Check this paper below - a C222(1) space group (a=212, b= 300, c=575) frequently appearing as a merohedral twin P2(1) with apparent C222(1) symmetry was exactly a major problem in the H. marismortui 50S structure determination. PoulBan N, Nissen P, Hansen J, Capel M, Moore PB, Steitz TA.Placement o

Re: [ccp4bb] the worst molecular replacment probes - three months

>> replacement search probes they used to get the correct solution purely >> with MR? >> >> perhaps in terms of %-scattering, RMSD, Z-score, LLG, or other possibly >> specific scoring values. >> >> -bryan >> > --

Re: [ccp4bb] the worst molecular replacment probes

possibly > specific scoring values. > > -bryan > > -- ======== Poul Nissen, professor, ph.d. Centre for Membrane Pumps in Cells and Disease - PUMPKIN University of Aarhus, Dept. Molecular Biology Gustav Wieds Vej 10C, DK

Re: [ccp4bb] Structures with pseudo-uridine?

Hi Yuan, Basically all tRNA structures in pdb will contain pseudo-U, and starting from old tRNAPhe literature you will find a lot on the structural consequences of having pseudo-U in RNA. Poul On 27/04/2007, at 3.45, Yuan Lin wrote: Dear All, I solved a structure of a pseudouridylated

Re: [ccp4bb] Highest shell standards

>> _http://xray.bmc.uu.se/gerard/gmrp/gmrp.html_ >> >> "To decide at which shell to cut off the resolution, we nowadays tend >> to use the following criteria for the highest shell: completeness > 80 >> %, multiplicity > 2, more than 60 % of the reflection